Quinone methide analog signal amplification

ABSTRACT

Disclosed herein are novel quinone methide analog precursors and embodiments of a method and a kit of using the same for detecting one or more targets in a biological sample. The method of detection comprises contacting the sample with a detection probe, then contacting the sample with a labeling conjugate that comprises an enzyme. The enzyme interacts with a quinone methide analog precursor comprising a detectable label, forming a reactive quinone methide analog, which binds to the biological sample proximally to or directly on the target. The detectable label is then detected. In some embodiments, multiple targets can be detected by multiple quinone methide analog precursors interacting with different enzymes without the need for an enzyme deactivation step.

CROSS REFERENCE TO RELATED APPLICATIONS

This patent application is a continuation of U.S. patent applicationSer. No. 16/183,583 filed on Nov. 7, 2018, which application is acontinuation of U.S. patent application Ser. No. 15/246,430 filed onAug. 24, 2016, which is a continuation of International Application No.PCT/EP2015/053556 filed Feb. 20, 2015, which claims priority to and thebenefit of U.S. Provisional Patent Application No. 61/943,940 filed Feb.24, 2014. Each patent application is incorporated herein by reference asif set forth in its entirety.

FIELD

This disclosure concerns novel quinone methide analog precursors andembodiments of a method and a kit comprising the same.

BACKGROUND

Immunohistochemistry (IHC) refers to the processes of detecting,localizing, and/or quantifying antigens, such as a protein, in abiological sample using specific binding moieties, such as antibodiesspecific to the particular antigens. IHC provides the substantialadvantage of identifying exactly where a particular protein is locatedwithin the tissue sample. It is also an effective way to examine thetissues themselves. In situ hybridization (ISH) refers to the process ofdetecting, localizing, and quantifying nucleic acids. Both IHC and ISHcan be performed on various biological samples, such as tissue (e.g.fresh frozen, formalin fixed, paraffin embedded) and cytologicalsamples. Recognition of the targets can be detected using various labels(e.g., chromogenic, fluorescent, luminescent, radiometric), irrespectiveof whether the target is a nucleic acid or an antigen. To robustlydetect, locate, and quantify targets in a clinical setting,amplification of the recognition event is desirable as the ability toconfidently detect cellular markers of low abundance becomesincreasingly important for diagnostic purposes. For example, depositingat the marker's sites hundreds or thousands of label molecules inresponse to a single antigen detection event enhances, throughamplification, the ability to detect that recognition event.

Adverse events often accompany amplification, such as non-specificsignals that are apparent as an increased background signal. Anincreased background signal interferes with the clinical analysis byobscuring faint signals that may be associated with low, but clinicallysignificant, expressions. Accordingly, while amplification ofrecognition events is desirable, amplification methods that do not theincrease background signal are highly desirable. One such method isTyramide Signal Amplification (TSA), which has also been referred to ascatalyzed reporter deposition (CARD). U.S. Pat. No. 5,583,001 disclosesa method for detecting and/or quantitating an analyte using ananalyte-dependent enzyme activation system that relies on catalyzedreporter deposition to amplify the detectable label signal. Catalysis ofan enzyme in a CARD or TSA method is enhanced by reacting a labeledphenol molecule with an enzyme. Modern methods utilizing TSA effectivelyincrease the signals obtained from IHC and ISH assays while notproducing significant background signal amplification (see, for example,U.S. application publication No. 2012/0171668 which is herebyincorporated by reference in its entirety for disclosure related totyramide amplification reagents). Reagents for these amplificationapproaches are being applied to clinically important targets to providerobust diagnostic capabilities previously unattainable (OPTIVIEW®Amplification Kit, Ventana Medical Systems, Tucson AZ, Catalog No.760-099).

TSA takes advantage of the reaction between horseradish peroxidase (HRP)and tyramide. In the presence of H₂O₂, tyramide is converted to ahighly-reactive and short-lived radical intermediate that reactspreferentially with electron-rich amino acid residues on proteins.Covalently-bound detectable labels can then be detected by variety ofchromogenic visualization techniques and/or by fluorescence microscopy.In solid-phase immunoassays such as IHC and ISH, where spatial andmorphological context is highly valued, the short lifetime of theradical intermediate results in covalent binding of the tyramide toproteins on tissue in close proximity to the site of generation, givingdiscrete and specific signal. While CARD broadly defines the use of ananalyte-dependent reporter enzyme (ADRE) to catalyze covalent binding ofnumerous detectable labels to proteins, HRP-based TSA is a commerciallyvalidated approach. No alternative ADRE systems exist despite a strongneed in the field for alternative amplification systems.

U.S. Pat. No. 7,291,474 to Bobrow postulates using hydrolase-based CARD.In particular, Bobrow hypothesizes that the activity probes2-difluoromethylphenyl and p-hydroxymandelic acid could be used asamplification reagents. The use of 2-difluoromethylphenyl andp-hydroxymandelic acid was described by Zhu et al., (2003) TetrahedronLetters, 44, 2669-2672; Lo et al., (2002) J. Proteome Res., 1, 35-40;Cesaro-Tadic et al., (2003) Nature Biotechnology, 21, 679-685; Janda etal., (1997) Science 275, 945-948; Halazy et al., (1990) BioorganicChemistry 18, 330-344; and Betley et al., (2002) Angew. Chem. Int. Ed.41, 775-777. Bobrow's suggested structures included the following:

wherein Y is a moiety capable of being cleaved by a hydrolytic enzyme; Lis a detectable label; X is a linking group; Z is a halogen; and R ishydrogen, alkyl, or halogen. In specific embodiments, R is hydrogen andthe Z groups are fluorine. These structures are generalizations of theparticular structures disclosed by Zhu et al., (2003) TetrahedronLetters, 44, 2669-2672. In particular, Zhu et al. describes thefollowing structures as known phosphatase inhibitors:

Based on these phosphatase inhibitors, Zhu et al. developed thefollowing activity probes:

Zhu discloses that the activity-based profiling of proteins is a provenand powerful tool in proteomic studies, whereby subclasses of enzymaticproteins can be selectively identified. As such, Zhu developed theactivity probes to signal the presence of active phosphatase enzymes.Zhu's strategy takes advantage of specific probes that react withdifferent classes of enzymes, leading to the formation of covalentprobe-protein complexes that are readily distinguished from othernon-reactive proteins in a crude proteome mixture.

Zhu et al. state that it was known that 2-difluoromethylphenyl phosphatewas a general phosphatase inhibitor against a broad spectrum ofdifferent phosphatases, including acid and alkaline phosphatases.Inhibition occurs as the phosphatases catalyze phosphate group cleavageto generate a reactive intermediary after a fluoride ion leaves. Thereactive intermediary reacts with the enzyme's active site to covalentlybind a fluorophore to the enzyme active site. But in doing so, it alsoinhibits the enzyme's ability to further hydrolytically cleavephosphates.

Using enzyme inhibitors in an amplification scheme to covalently bindsignal generating moieties to a substrate is understood to beself-limiting as the generation of bound signal can destroy the activityof the enzyme. There has been a recognition in the art that pursuingenhancements made to these reagents would likely be self-defeating asthe improved performance (e.g. turnover, specificity) would result inmore efficient destruction of the enzyme's active site. Thus, in orderto get signal amplification by binding multiple signal generatingmoieties, multiple enzymes first have to be bound proximally to thetarget. Accordingly, the compounds disclosed by Zhu et al. and Bobrowhave never been developed into a commercially viable detection reagentfor an amplification system for IHC or ISH.

Furthermore, the amplification approaches described thus far enable thedeposition of fluorescent compounds. Fluorescence imaging is oftenimplemented because it is extraordinarily sensitive; the detection ofvery few fluorophore molecules is now routine. However, this sensitivityis achieved using dark-field imaging, which has certain pragmaticlimitations. For example, bright-field primary staining (e.g.,hematoxylin and eosin staining) cannot be concurrently observed, makingit more difficult to correlate fluorescent signal with morphologicalfeatures. It is well known that fluorescence-based detection isroutinely 1000 times more sensitive than absorbance-reflectance-basedapproaches (e.g. chromogenic-based detection). As such, a methodologyappropriate for fluorescence detection would require a 1000-foldimprovement for use as a chromogenic detection methodology. Increasingthe performance of an enzyme-based detection system by 1000-fold isnon-trivial. To date, only tyramide-based systems have achieved thisincreased performance.

While robust reagents are available, a need persists for alternativesignal amplification approaches that produce robust amplificationwithout increasing background signals. Moreover, methods for amplifyingthe detection of two or more distinct targets in a tissue sample aredesirable.

SUMMARY

The quinone methide analog precursors (QMPs) and embodiments for usingthese QMPs disclosed herein provide substantially superior results tothose disclosed in the prior art. The QMPs separate the detectable labelfunction from the quinone methide generation and nucleophilestabilization functions within the molecule. Also disclosed herein areembodiments of a method for utilizing QMPs for IHC and/or ISH stainingin tissue, such as formalin-fixed, paraffin-embedded (FFPE) tissue. Tothe inventors' knowledge, this has not been successfully demonstratedbefore. Embodiments of the method of using QMPs for amplifying thedetection of one or more distinct targets in a tissue sample provideimproved signal quality and reduced off-target staining, compared topreviously known, non-QMP methods. When the disclosed method is used todetect multiple targets, either simultaneously or sequentially, thetargets can be detected by chromogenic- or fluorescence-based detectionmethods, or a combination thereof.

In some embodiments, a QMP has a formula

or a salt or solvate thereof, where Z is O, S or NR^(a) and R¹ is anenzyme recognition group, or ZR¹ is an enzyme recognition group; R¹ is—C(LG)(R⁵)(R³R⁴), —R³R⁴ or —C(LG)(R⁵)(R⁶); R⁹, R¹¹ and R¹² are eachindependently hydrogen, halo, cyano, aliphatic, alkoxy, NO₂, N(R^(c))₂,aryl, haloalkyl, —C(O)alkyl, —C(S)alkyl, —C(O)OH, —C(O)Oalkyl,—C(O)NHR^(c), —C(O)N(R^(c))₂, —R³R⁴ or two adjacent groups together forman aliphatic ring or aryl ring; and R¹⁰ is hydrogen, halo, cyano,aliphatic, alkoxy, NO₂, N(R^(c))₂, aryl, haloalkyl, —C(O)alkyl,—C(S)alkyl, —C(O)OH, —C(O)Oalkyl, —C(O)NHR^(c), —C(O)N(R^(c))₂, —R³R⁴,—C(LG)(R⁵)(R⁶) or with one of R⁹ or R¹¹ form an aliphatic ring or arylring.

Also with reference to the formula, LG is a leaving group, or ZR¹ and LGtogether form a phosphodiester; R³ is a linker or a bond; R⁴ is adetectable label; each R⁵ is independently hydrogen, halo, cyano, loweralkyl, lower haloalkyl, —C(O)alkyl, —C(S)alkyl, —C(O)OH, —C(O)Oalkyl,—C(O)NHR^(c) or —C(O)N(R^(c))₂; each R⁶ is independently hydrogen, halo,cyano, lower alkyl, lower haloalkyl, —C(O)alkyl, —C(S)alkyl, —C(O)OH,—C(O)Oalkyl, —C(O)NHR^(c) or —C(O)N(R^(c))₂; R^(a) is hydrogen oraliphatic; and each R^(c) independently is hydrogen, aryl, aliphatic orheteroaliphatic, or two R^(c) moieties together form a heteroaliphaticring. Additionally, at least one of R⁸ and R¹⁰ comprises LG, and atleast one of R⁸ and R¹⁰ comprises or consists of R³R⁴, and if LG ishalo, then R⁵ and R⁶ are not halo.

In certain embodiments, the QMP has a formula selected from

In some embodiments, R¹ or ZR¹ is a phosphate, amide, nitro, urea,sulfate, methyl, ester, beta-lactam or sugar. Z may be O and/or ZR¹ maybe —OP(O)(OH)₂, NO₂, —NHC(O)R, —OC(O)CH₃, —OC(O)CH₂CH₃, —NHC(O)NH₂,—OS(O)₂OH, OCH₃ or a salt thereof. In some embodiments, the sugar isα-glucose, β-glucose, α-galactose, β-galactose, α-glucuronose orβ-glucuronose.

LG may be any suitable leaving group, such as a halide, sulfate ester,carboxylate, inorganic ester, thiolate, amine, aryloxy, alkoxy, orheteroaryl. In some embodiments, LG is fluoride, chloride, azide,acetate, methoxy, ethoxy, isopropoxy, phenoxide, —OS(O)₂CH₃,—OS(O)₂C₆H₄CH₃, —OS(O)₂C₆H₅, —OS(O)₂C₆H₄CX₃, —OC₆H₅, —N₂ ⁺, —NH₃ ⁺,—NC₅H₅ ⁺, —O-alkyl, —OC(O)alkyl, —OC(O)H, —N(R^(b))₃ ⁺ or1,4-diazabicyclo[2.2.2]octane (DABCO), where each X independently isfluoro, chloro, bromo or iodo, and each R^(b) independently is hydrogen,or lower alkyl, or two R^(b) moieties together form a heteroaliphaticring. In certain embodiments, LG is F.

Certain disclosed method embodiments comprise contacting a biologicalsample with a first detection probe specific to a first target. Thebiological sample is contacted with a first labeling conjugatecomprising a first enzyme. The biological sample is also contacted witha first QMP comprising a first enzyme recognition group and a firstdetectable label. The first enzyme cleaves the first enzyme recognitiongroup, thereby converting the first QMP into a first reactive quinonemethide analog (QM), which covalently binds to the biological sampleproximally to or directly on the first target. Contacting the biologicalsample comprises (i) contacting the biological sample with the first QMPat a precursor concentration, effective to give a desired level ofamplification, such as a concentration from greater than zero to 1 mM;(ii) contacting the biological sample with the first QMP at a pHeffective to reduce diffusion and/or off-target staining to a desiredamount, such as a pH from greater than 7 to 14, or from 8 to 12; (iii)contacting the biological sample with the first QMP in the presence of asalt, such as magnesium chloride, at a salt concentration effective toreduce diffusion and/or off-target staining to a desired amount,typically from 0.1 M to 2 M, or from 0.5 M to 1.25 M; (iv) contactingthe biological sample with a compound disclosed herein; or (v) anycombination thereof. The first target is then detected by detecting thefirst detectable label. The method may be an automated process.

The precursor concentration may be from 50 μM to 500 μM for chromogenicstaining, or from 50 nM to 10 μM for fluorescent staining and haptenamplification.

In some examples, the first labeling conjugate comprises an antibodycoupled to the first enzyme. The antibody may be an anti-species or ananti-hapten antibody. The first labeling conjugate may be associated,either directly or indirectly, with the first detection probe. The firstdetection probe may comprise a hapten or an anti-species antibody andthe first labeling probe comprise a corresponding anti-hapten or asecond anti-species antibody. The first enzyme and first enzymerecognition group may be any suitable enzyme and enzyme recognitiongroup that will interact to form a QM.

The first reactive QM reacts with a nucleophile within the biologicalsample, the first labeling conjugate, the first detection probe, orcombinations thereof. Typical nucleophiles comprise an amino,sulfhydryl, or hydroxyl group on an amino acid, nucleic acid residue orcarbohydrate.

The first QMP may have a formula as disclosed above, or alternatively, aformula selected from

or a salt or solvate thereof. With respect to these formulas, Z, LG,R^(a), R^(c), R¹, R³, R⁴, R⁵, R⁶ and R⁹-R¹² are as previously defined,R¹³-R²⁹ are each independently defined as for R⁹, R¹¹ and R¹², and atleast one of R⁹-R²⁹ comprises or consists of R³R⁴.

The method may be a multiplexed method. In some embodiments, in additionto detecting a first target the method further comprises contacting thebiological sample with a second binding moiety specific to a secondtarget. The second target is labeled with a second enzyme through thesecond binding moiety. The biological sample is contacted with a seconddetection precursor compound that interacts with the second enzyme todeposit a second detection compound directly on or proximally to thesecond target. The second detection compound is then detected. The firstenzyme and second enzyme typically are different enzymes. Contacting thefirst and second targets with the respective binding moieties and/ordetecting the first and second detection compounds may occursequentially or substantially contemporaneously.

In certain embodiments, the first enzyme reacts selectively with thefirst QMP, and the second enzyme reacts selectively with the seconddetection precursor compound. In particular embodiments, the firstenzyme is an alkaline phosphatase and the first enzyme recognition groupis a phosphate. The second enzyme may be a peroxidase.

In some embodiments, the second detection precursor compound is a secondQMP comprising a second enzyme recognition group and a second detectablelabel. The second QMP interacts with the second enzyme to form a secondQM that covalently binds to the biological sample proximally to ordirectly on the second target. The second enzyme typically is adifferent enzyme than the first, such as a β-galactosidase where thesecond enzyme recognition group is a β-galactoside.

A person of ordinary skill in the art will understand that the methodcan be expanded to include detecting additional distinct targets. Thiscan be achieved by contacting the biological sample with additionalbinding moieties specific to the targets, labeling the binding moietieswith different enzymes, contacting the sample with detection precursorcompounds selected for the enzymes and detecting the detectioncompounds.

A kit comprising a staining amplification compound disclosed herein isalso disclosed. In some embodiments, the kit comprises anenzyme-antibody conjugate, a QMP, a solvent mixture, and a pH adjustsolution. The solvent mixture may comprise an organic solvent and anaqueous buffer. In some embodiments, the organic solvent is DMSO. Theaqueous buffer may have a pH range of from pH 0 to pH 5 or from pH 1 topH 3. In some embodiments, the pH adjust solution has a pH range of frompH 8 to pH 12. In particular embodiments, the kit includes a salt, suchas magnesium chloride, which may have a concentration of from 0.25 M to1.5 M. In some embodiments, the QMP is a compound disclosed herein, thesolvent mixture comprises DMSO and a glycine buffer at pH 2, the pHadjust solution is a Tris buffer with a pH range of from pH 8 to pH 10,and/or the kit comprises further comprising magnesium chloride at aconcentration of from 0.5 M to 1.25 M.

The foregoing and other objects, features, and advantages of theinvention will become more apparent from the following detaileddescription, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor.

Copies of this patent or patent application publication with colordrawing(s) will be provided by the Office upon request and payment ofthe necessary fee.

FIG. 1 is a schematic diagram illustrating detecting a target using aQMP comprising a detectable label.

FIG. 2(A) is a table providing exemplary QMPs.

FIG. 2(B) illustrates the structures of exemplary QMPs.

FIG. 3(A) illustrates phosphatase-mediated conversion of a QMP withdetectable label to a quinone methide that amplifies a target signal.

FIG. 3(B) is an additional illustration of phosphatase-mediatedconversion of a QMP with detectable label to a quinone methide thatamplifies a target signal.

FIG. 3(C) is an additional illustration of phosphatase-mediatedconversion of a QMP with detectable label to a quinone methide thatamplifies a target signal.

FIG. 4 illustrates one exemplary embodiment of a method for amplifyingtarget detection in biological tissue.

FIG. 5 illustrates a second exemplary embodiment of a method foramplifying target detection in biological tissue.

FIG. 6(A) is a microphotograph illustrating the increase in stainingintensity of QMP-Dabsyl derivatives with PEG linkers, for Bcl-6 ontonsil tissue at 20× magnification using phosphate-QMP-Dabsyl (250 uM).

FIG. 6(B) is a microphotograph illustrating the increase in stainingintensity of QMP-Dabsyl derivatives with PEG linkers, for Bcl-6 ontonsil tissue at 20× magnification using phosphate-QMP-PEG₄-Dabsyl (250uM).

FIG. 6(C) is a microphotograph illustrating the increase in stainingintensity of QMP-Dabsyl derivatives with PEG linkers, for Bcl-6 ontonsil tissue at 20× magnification using phosphate-QMP-PEG₈-Dabsyl (250uM).

FIG. 7(A) is a microphotograph illustrating the increase in stainingintensity of QMP-Tamra derivatives with PEG linkers, for Bcl-6 on tonsiltissue at 20× magnification using phosphate-QMP-Tamra (250 uM).

FIG. 7(B) is a microphotograph illustrating the increase in stainingintensity of QMP-Tamra derivatives with PEG linkers, for Bcl-6 on tonsiltissue at 20× magnification using phosphate-QMP-PEG₄-Tamra (250 uM).

FIG. 7(C) is a microphotograph illustrating the increase in stainingintensity of QMP-Tamra derivatives with PEG linkers, for Bcl-6 on tonsiltissue at 20× magnification using phosphate-QMP-PEG₈-Tamra (250 uM).

FIG. 8(A) is a microphotograph from a duplex brightfield IHC assay ofbreast tissue at 10× magnification, illustrating simultaneous antibodyincubation and sequential chromogenic detection of Pan-Keratin(QM-PEG8-Dabsyl, yellow) and Her2 (Tyr-TAMRA, purple).

FIG. 8(B) is a portion of FIG. 8(A) magnified to 40× magnification.

FIG. 9 is a microphotograph from a quadruplex brightfield IHC assay oftonsil tissue at 40× magnification, illustrating sequential detection ofCD8 (Tyr-Rhodamine-110, maroon), CD3 (QM-Cy5, blue), FoxP3 (Tyr-Tamra,purple) and Pan-keratin (QM-PEG8-Dabsyl, yellow).

FIG. 10 is a microphotograph of a fluorescent duplex assay utilizingboth AP-based QMP (Ki67, dark red, nuclear) and HRP-based TSA (Bcl2,light green, membrane) detections on FFPE tonsil tissue.

FIG. 11 is a microphotograph of quinone methide staining of E-cadherinon breast tissue at pH 7.5.

FIG. 12 is a microphotograph of quinone methide staining of E-cadherinon breast tissue at pH 10.

FIG. 13(A) is a microphotograph illustrating functional stainingamplification of Ki67 on FFPE tonsil tissue by a QMP having a monofluoroleaving group and a 5-nitro-3-pyrazolecarbamide (nitropyrazole)detectable label.

FIG. 13(B) is a microphotograph illustrating a VENTANA ultraView DABcontrol of the assay illustrated in FIG. 13(A).

FIG. 14(A) is a microphotograph illustrating functional staining ofCD-10 on FFPE tonsil tissue by a QMP having a monofluoro leaving groupand a 5-nitro-3-pyrazolecarbamide (nitropyrazole, NP) detectable label.

FIG. 14(B) is a microphotograph illustrating functional staining ofamplification of CD-10 on FFPE tonsil tissue by a QMP having amonofluoro leaving group and a 5-nitro-3-pyrazolecarbamide(nitropyrazole, NP) detectable label, followed by an anti-NPantibody/alkaline phosphatase conjugate and fast red staining.

FIG. 14(C) is a microphotographs illustrating functional staining ofBcl2 on FFPE tonsil tissue by a QMP having a monofluoro leaving groupand a 5-nitro-3-pyrazolecarbamide (nitropyrazole, NP) detectable label,followed by an anti-NP antibody/alkaline phosphatase conjugate and fastred staining.

FIG. 14(D) is a microphotograph illustrating functional staining Her3 onFFPE breast tissue by a QMP having a monofluoro leaving group and a5-nitro-3-pyrazolecarbamide (nitropyrazole, NP) detectable label,followed by an anti-NP antibody/alkaline phosphatase conjugate and fastred staining.

FIG. 15(A) is a microphotograph illustrating functional stainingamplification of Bcl2 on FFPE tonsil tissue by a QMP having a monofluoroleaving group and a TAMRA detectable moiety.

FIG. 15(B) is a microphotograph illustrating functional stainingamplification of an AF700 detectable moiety.

FIG. 15(C) is a microphotograph illustrating functional stainingamplification of nitropyrazole detectable followed by a quantum dot(QD525)-labeled, anti-nitropyrazole antibody.

FIG. 16(A) is a microphotograph illustrating functional stainingamplification of Ki67 on FFPE tonsil tissue by a QMP having a monofluoroleaving group and a Dabsyl detectable moiety.

FIG. 16(B) is a microphotograph illustrating functional stainingamplification of Ki67 on FFPE tonsil tissue by a QMP having a TAMRAdetectable moiety.

FIG. 16(C) is a microphotograph illustrating functional stainingamplification of Ki67 on FFPE tonsil tissue by a QMP having a Cy5detectable moiety.

FIG. 16(D) is a microphotograph illustrating functional stainingamplification of Ki67 on FFPE tonsil tissue by a QMP having a Rhodamine110 detectable moiety.

FIG. 17(A) is a microphotograph illustrating functional stainingamplification of epidermal growth factor receptor (EGFR) informalin-fixed, paraffin-embedded (FFPE) skin tissue by ultraView3,3′-diaminobenzidine (DAB).

FIG. 17(B) is a microphotograph illustrating functional stainingamplification of epidermal growth factor receptor (EGFR) informalin-fixed, paraffin-embedded (FFPE) skin tissue utilizing a QMPhaving a difluoro leaving group and conjugated with a biotin detectablelabel.

FIG. 18 is a microphotographs illustrating AP-based CARD IHC (BCL6 onFFPE tonsil tissue) using a biotinylated difluoro QM precursor followedby DAB detection at varying concentrations of the QM precursor wherepanel A—DAB control, panel B—1 μM, panel C—10 μM and panel D—20 μM.

FIG. 19 is a microphotographs of AP-based CARD IHC (BCL6 on FFPE tonsiltissue) using 20 μM biotinylated difluoro QM precursor followed by DABdetection illustrating staining results with varying pH where panelA—DAB control; panel B pH=7.0; panel C pH=8.0; panel D pH=8.5; panel EpH=9.0; panel F pH=10.0; panel G pH=11.0; panel H pH=12.0.

FIG. 20 is a microphotographs of AP-based CARD IHC (BCL6 on FFPE tonsiltissue) using 250 nM biotinylated monofluoro QM precursor followed byDAB detection illustrating staining results with varying pH with panelA—DAB control; panel B—pH=7.0; panel C—pH=8.0; panel D—pH=8.5; panel EpH=9.0; panel F pH=10.0; panel G-pH=11.0; panel H—pH=12.0.

FIG. 21(A) is a microphotograph illustrating optimal stainingamplification of Ki67 on FFPE tonsil tissue by ultraView control.

FIG. 21(B) is a microphotograph illustrating optimal stainingamplification of Ki67 on FFPE tonsil tissue with a QMP having a pyridineleaving group and conjugated with a biotin detectable label.

FIG. 21(C) is a microphotograph illustrating optimal stainingamplification of Ki67 on FFPE tonsil tissue with a DABCO leaving groupand conjugated with a biotin detectable label.

FIG. 21(D) is a microphotograph illustrating optimal stainingamplification of Ki67 on FFPE tonsil tissue with a triethylamine leavinggroup and conjugated with a biotin detectable label.

FIG. 22(A) is a microphotograph illustrating optimal functional stainingamplification of Ki67 on FFPE tonsil tissue by ultraView DAB FIG. 22(B)is a microphotograph illustrating optimal functional stainingamplification of Ki67 on FFPE tonsil tissue with a QMP having amonofluoro leaving group with a biotin detectable label.

FIG. 22(C) is a microphotograph illustrating optimal functional stainingamplification of Ki67 on FFPE tonsil tissue with a QMP having an acetateleaving group.

FIG. 22(D) is a microphotograph illustrating optimal functional stainingamplification of Ki67 on FFPE tonsil tissue a methoxy leaving group anda biotin detectable label.

FIG. 23(A) is a microphotograph illustrating functional stainingamplification of Bcl2 on FFPE tonsil tissue by a QMP having a monofluoroleaving group, a biotin detectable label, and an aniline amide linker(Compound 7).

FIG. 23(B) is a microphotograph illustrating functional stainingamplification of Bcl2 on FFPE tonsil tissue by a QMP having a monofluoroleaving group, a biotin detectable label, and a benzoic amide linker(Compound 21).

FIG. 23(C) is a microphotograph illustrating functional stainingamplification of Bcl2 on FFPE tonsil tissue by a QMP having a monofluoroleaving group, a biotin detectable label, and a tyramide amide linker(Compound 14).

FIG. 24(A) is a further magnification of the microphotographs in FIG.17(B).

FIG. 24(B) is a further magnification of the microphotographs in FIG.17(B).

FIG. 25(A) is a further magnification of the microphotographs in FIG.17(A).

FIG. 25(B) is a further magnification of the microphotographs in FIG.17(A).

FIG. 26 illustrates the trapping the QM intermediate from amonofluroinated QM precursor with Tris.

FIG. 27 is a HPLC chromatograms of the compounds from the reactionillustrated in FIGS. 24(A)-(B), where panel A t=0 min., panel B t=10min., panel C t=10 min.

FIG. 28 provides exemplary structures of QMPs disclosed herein wherepanel A is a para-di-substituted QMP conjugated to a TAMRA, panel B is apara-di-substituted QMP conjugated to a Dabsyl, panel C is anortho-di-substituted QMP conjugated to a Dabsyl, and panel D is anortho-di-substituted QMP conjugated to a Cy5.

FIG. 29(A) is a microphotograph illustrating the improvement in stainingquality of CD8 on tonsil tissue using 0.125 M magnesium chloride.

FIG. 29(B) is a microphotograph illustrating the improvement in stainingquality of CD8 on tonsil tissue using 1.05 M magnesium chloride.

FIG. 30(A) is a microphotograph illustrating functional staining ofKi-67 on tonsil tissue at 20× magnification with β-galactosidase enzymeand with β-galactoside-QMP-Cy5 (125 uM) and Nuclear Fast Red CS.

FIG. 30(B) is a microphotograph illustrating functional staining ofKi-67 on tonsil tissue at 20× magnification with β-galactosidase enzymeand with β-galactoside-QMP-Cy5 (125 uM) and Hematoxylin CS.

FIG. 30(C) is a microphotograph illustrating functional staining ofKi-67 on tonsil tissue at 20× magnification with β-galactosidase enzymeand with β-galactoside-QMP-Cy3 (100 uM) and Hematoxylin CS.

FIG. 31 is a microphotograph from a duplex brightfield IHC assay,illustrating simultaneous detection of Bcl-6 (Phospho-QM-PEG8-Dabsyl,yellow) and Ki67 (β-Gal-QM-Cy5, blue) on tonsil tissue with ahematoxylin counterstain, using simultaneous antibody incubation andsimultaneous chromogenic detection.

FIG. 32(A) is a microphotograph from a triplex brightfield IHC assay,illustrating simultaneous detection of Her2, ER, PR on breast tissuewith Phospho-QM-PEG8-Dabsyl, β-Gal-QM-Cy5, Tyr-Tamra, with a hematoxylincounterstain, using simultaneous antibody incubation and simultaneouschromogenic detection.

FIG. 32(B) is a microphotograph from a triplex brightfield IHC assay,illustrating simultaneous detection of Her2, ER, PR on breast tissuewith Phospho-QM-PEG8-Dabsyl, β-Gal-QM-Cy5, Tyr-Tamra, with a hematoxylincounterstain, using simultaneous antibody incubation and simultaneouschromogenic detection.

FIG. 33 is a microphotograph from a quadruplex brightfield IHC assay,illustrating sequential detection of Her2 (HRP DAB, brown), PR(0-Gal-QM-Cy5, blue), ER (Tyr-Tamra, purple) and Ki67(Phospho-QM-PEG8-Dabsyl, yellow) on breast tissue.

FIG. 34 is a second microphotograph from a quadruplex brightfield IHCassay, illustrating sequential detection of Her2 (HRP DAB, brown), PR(0-Gal-QM-Cy5, blue), ER (Tyr-Tamra, purple) and Ki67(Phospho-QM-PEG8-Dabsyl, yellow) on breast tissue.

FIG. 35(A) is a microphotograph of chromosome 17 centromere ISH ontonsil tissue with QM-green (PEG8-Dabsyl and Cy5).

FIG. 35(B) MCF-7 is a microphotograph of chromosome 17 centromere ISH onxenografts with QM-green (PEG8-Dabsyl and Cy5).

FIG. 36 ) is a microphotograph of four different staining protocolsshowing the same biomarkers (panel A—Her2, panel B—Ki-67, panel C—ER andpanel C—PR) on FFPE breast tissue at 40× magnification, stained bysequential detection using two HRP based detections and two AP QMP baseddetection systems.

FIG. 37 is a microphotograph of different staining protocols (panels A-Bor panels C-D) of panel A—CD3, panel B—CD8, panel C—CD20 (or CD68) andpanel D-FoxP3 at 5× magnification on FFPE tonsil tissue, stained bysequential detection using two HRP based detections and two AP QMP baseddetection systems.

FIG. 38(A) is a microphotograph illustrating functional stainingamplification of E-cadherin on FFPE breast tissue by an ortho- andpara-QMP-Tamra at 10× magnification using compound 36.

FIG. 38(B) is a microphotograph illustrating functional stainingamplification of E-cadherin on FFPE breast tissue by an ortho- andpara-QMP-Tamra at 10× magnification using compound 28.

DETAILED DESCRIPTION I. Definitions

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes VII, published by Oxford UniversityPress, 2000 (ISBN 019879276X); Kendrew et al. (eds.), The Encyclopediaof Molecular Biology, published by Blackwell Publishers, 1994 (ISBN0632021829); and Robert A. Meyers (ed.), Molecular Biology andBiotechnology: a Comprehensive Desk Reference, published by Wiley, John& Sons, Inc., 1995 (ISBN 0471186341); and other similar references.

As used herein, the singular terms “a,” “an,” and “the” include pluralreferents unless context clearly indicates otherwise. Similarly, theword “or” is intended to include “and” unless the context clearlyindicates otherwise. Also, as used herein, the term “comprises” means“includes.” Hence “comprising A or B” means including A, B, or A and B.It is further to be understood that all nucleotide sizes or amino acidsizes, and all molecular weight or molecular mass values, given fornucleic acids or polypeptides or other compounds are approximate, andare provided for description. Although methods and materials similar orequivalent to those described herein can be used in the practice ortesting of the present disclosure, suitable methods and materials aredescribed below. All publications, patent applications, patents, andother references mentioned herein are incorporated by reference in theirentirety. In case of conflict, the present specification, includingexplanations of terms, will control. In addition, the materials,methods, and examples are illustrative only and not intended to belimiting.

Unless otherwise indicated, all numbers expressing quantities ofcomponents, molecular weights, percentages, temperatures, times,concentrations, and so forth, as used in the specification or claims areto be understood as being modified by the term “about.” Accordingly,unless otherwise indicated, implicitly or explicitly, the numericalparameters set forth are approximations that may depend on the desiredproperties sought and/or limits of detection under standard testconditions/methods. When directly and explicitly distinguishingembodiments from discussed prior art, the embodiment numbers are notapproximates unless the word “about” is recited.

For the general formulas provided below, if no substituent is indicated,a person of ordinary skill in the art will appreciate that thesubstituent is hydrogen. A bond that is not connected to an atom, but isshown, for example, extending to the interior of a ring system,indicates that the position of such substituent is variable. A curvedline drawn through a bond indicates that some additional structure isbonded to that position. Moreover, if no stereochemistry is indicatedfor compounds having one or more chiral centers, all enantiomers anddiastereomers are included. Similarly, for a recitation of aliphatic oralkyl groups, all structural isomers thereof also are included.

In order to facilitate review of the various embodiments of thedisclosure, the following explanations of specific terms are provided:

Aliphatic: A substantially hydrocarbon-based compound, or a radicalthereof (e.g., C₆H₁₃, for a hexane radical), including alkanes, alkenes,alkynes, including cyclic versions thereof, and further includingstraight- and branched-chain arrangements, and all stereo and positionisomers as well. Unless expressly stated otherwise, an aliphatic groupcontains from one to twenty-five carbon atoms; for example, from one tofifteen, from one to ten, from one to six, or from one to four carbonatoms. The term “lower aliphatic” refers to an aliphatic groupcontaining 1-10 carbon atoms. Unless expressly referred to as an“unsubstituted aliphatic,” an aliphatic group can either beunsubstituted or substituted.

Alkyl: A hydrocarbon group having a saturated carbon chain. The chainmay be cyclic, branched or unbranched. The term lower alkyl means thechain includes 1-10 carbon atoms. Unless otherwise stated, an alkylgroup may be substituted or unsubstituted.

Alkoxy: A group having a formula —O-alkyl, where alkyl is as definedherein.

Analog: An analog is a molecule that differs in chemical structure froma parent compound, for example a homolog (differing by an increment inthe chemical structure, such as a difference in the length of an alkylchain), a molecular fragment, a structure that differs by one or morefunctional groups, a change in ionization. Structural analogs are oftenfound using quantitative structure activity relationships (QSAR), withtechniques such as those disclosed in Remington (The Science andPractice of Pharmacology, 19th Edition (1995), chapter 28).

Aromatic or aryl: An aromatic carbocyclic or heterocyclic group of,unless specified otherwise, from 6 to 15 ring atoms having a single ring(e.g., phenyl, pyridyl) or multiple condensed rings in which at leastone ring is aromatic (e.g., quinoline, indole, benzodioxole, and thelike), provided that the point of attachment is through an atom of anaromatic portion of the aryl group. If any aromatic ring portioncontains a heteroatom, the group is a heteroaryl, otherwise the group isa carbocyclic aryl group. Aryl groups may be monocyclic, bicyclic,tricyclic or tetracyclic. Unless otherwise stated, an aryl group may besubstituted or unsubstituted.

Aryloxy: A group having a formula —O-aryl, where aryl is as definedherein.

Conjugate: Two or more moieties directly or indirectly coupled together.For example, a first moiety may be covalently or noncovalently (e.g.,electrostatically) coupled to a second moiety. Indirect attachment ispossible, such as by using a “linker” (a molecule or group of atomspositioned between two moieties).

Conjugated system: As used herein, the term “conjugated system” refersto a compound including overlapping orbitals (typically p-orbitals) withdelocalized pi electrons. Typically the compound includes alternatingsingle and multiple bonds. The overlapping p-orbitals bridge the singlebonds between adjacent overlapping p-orbitals. Lone pairs, radicals, andcarbenium ions may be part of the system. The system may be cyclic,acyclic, or a combination thereof. Exemplary conjugated systems include,but are not limited to aromatic compounds such as benzene, pyrazole,imidazole, pyridine, pyrimidine, pyrrole, furan, thiophene, naphthalene,anthracene, indole, benzoxazole, benzimidazole, and purine.

Contacting: Placement that allows association between two or moremoieties, particularly direct physical association, for example both insolid form and/or in liquid form (for example, the placement of abiological sample, such as a biological sample affixed to a slide, incontact with a composition, such as a solution containing thecompositions disclosed herein).

Detect: To determine if an agent (such as a signal or particularantigen, protein or nucleic acid) is present or absent, for example, ina sample. In some examples, this can further include quantification,and/or localization, for example localization within a cell orparticular cellular compartment. “Detecting” refers to any method ofdetermining if something exists, or does not exist, such as determiningif a target molecule is present in a biological sample. For example,“detecting” can include using a visual or a mechanical device todetermine if a sample displays a specific characteristic. In certainexamples, light microscopy and other microscopic means are used todetect a detectable label bound to or proximally to a target.

Detectable Label: A molecule or material that can produce a detectable(such as visually, electronically or otherwise) signal that indicatesthe presence and/or concentration of a target, such as a targetmolecule, in a sample, such as a tissue sample. When conjugated to amolecule capable of binding directly or proximally to a target, thedetectable label can be used to locate and/or quantify the target.Thereby, the presence and/or concentration of the target in a sample canbe detected by detecting the signal produced by the detectable label. Adetectable label can be detected directly or indirectly, and severaldifferent detectable labels conjugated to different molecules can beused in combination to detect one or more targets. Multiple detectablelabels that can be separately detected can be conjugated to differentmolecules that bind directly or proximally to different targets toprovide a multiplexed assay that can provide detection of the multipletargets in a sample. As used herein, detectable labels include colored,fluorescent, phosphorescent, and luminescent molecules, and haptens.

Electron donating group: An atom or functional group capable of donatingsome of its electron density into a conjugated system. Electron densitycan be donated through a bonds (inductive) or through π bonds(resonance). Some functional groups are donating groups by one mechanismand withdrawing groups through the other mechanism. Exemplary electrondonating groups include, but are not limited to, —NH₂, —NHR, —NR₂, —OH,—CH═CH₂, —NHC(O)R, —OR, —R, where R is alkyl, such as lower alkyl (e.g.,methyl, ethyl).

Electron withdrawing group: An atom or functional group capable ofwithdrawing electron density from a conjugated system. Electron densitycan be withdrawn through a bonds (inductive) or through π bonds(resonance). Some functional groups are donating groups by one mechanismand withdrawing groups through the other mechanism. Exemplary electronwithdrawing groups include, but are not limited to, halo, haloalkyl,—NH₃ ⁺, —NO₂, —CH═CH₂, —CN, —SO₃H, —C(O)OH, —C(O)H, —C(O)R, —CN,—C(O)OR, —NR₃ ⁺, where R is alkyl, such as lower alkyl (e.g., methyl,ethyl).

Heteroaliphatic: An aliphatic compound where one or more carbon has beenreplaced with a heteroatom. Exemplary heteroatoms include, but are notlimited to, O, S, N, P, Si or B. Heteroaliphatic moieties may besubstituted or unsubstituted. Substitution may be at a carbon atom or ata heteroatom.

Heteroaryl: An aromatic compound or group having at least oneheteroatom, i.e., one or more carbon atoms in the ring has been replacedwith an atom having at least one lone pair of electrons, typicallynitrogen, oxygen, phosphorus, silicon, or sulfur. Unless otherwisestated, a heteroaryl group may be substituted or unsubstituted.

Inorganic ester: An ester derived from an inorganic acid and an alcohol.Exemplary inorganic acids include, but are not limited to, phosphoricacid, sulfuric acid, nitric acid or boric acid. Inorganic estersinclude, but are not limited to, sulfates, phosphates, nitrates orborates, for example, triphenyl phosphate.

Leaving group: A molecular fragment that is eliminated with a pair ofelectrons during heterolytic bond cleavage. Another term for leavinggroup is nucleofuge. Leaving groups may be anions or neutral molecules(if a leaving group is positively charged while bound to the molecule,it will become neutral when it leaves with a pair of electrons). Theability of a molecular fragment to be a leaving group (i.e., itsnucleofugality or nucleofugacity) is correlated with its stability. Insome circumstances, e.g., when the leaving group is a weak base, theability of a leaving group to depart may be related to the pK_(a) of theleaving group's conjugate acid, with lower pK_(a) often but not alwaysbeing correlated with better leaving group ability. A person of ordinaryskill in the art is aware of readily available tables, e.g., in organicchemistry textbooks, that indicate the relative nucleofugality ofleaving groups.

Substantially non-inhibiting: A QMP is substantially non-inhibiting ifit forms a quinone methide that so diffuses from the enzyme as to notreact with the reactive site of the enzyme. Substantially non-inhibitingcan be established by functionally testing a particular enzyme and QMP.Generally, if staining, as described herein, increases over extendedperiods of time (e.g. >5 minutes), the QMP is substantiallynon-inhibiting. If a QMP inhibits the enzyme, the amount of stainingwill not increase over time or with the addition of more QMP.

Nucleophile: A chemical species capable of donating an electron pair toa positively-charged (or partially positive) atom to form a chemicalbond during a chemical reaction. Anions and molecules with a lone pairof electrons or at least one pi bond can act as nucleophiles.

Oligonucleotide: A plurality of joined nucleotides joined byphosphodiester bonds, between about 6 and about 300 nucleotides inlength. As used herein, the term oligonucleotide refers to DNAoligonucleotides, RNA oligonucleotides, synthetic oligonucleotides(e.g., non-naturally occurring DNA or RNA sequences), andoligonucleotide analogs. An oligonucleotide analog refers to moietiesthat function similarly to oligonucleotides but have non-naturallyoccurring portions. For example, oligonucleotide analogs can containnon-naturally occurring portions, such as altered sugar moieties orinter-sugar linkages, such as a phosphorothioate oligodeoxynucleotide.Functional analogs of naturally occurring polynucleotides can bind toRNA or DNA, and include peptide nucleic acid molecules.

Probe: A substance used to detect or identify another substance in asample. As used herein, a probe may be an antibody, an antibodyfragment, an isolated nucleic acid, or an isolated syntheticoligonucleotide capable of specifically binding to a desired target,e.g., a target protein or nucleic acid sequence present in a tissuesample. The probe may comprise a detectable label or reporter molecule(e.g., a hapten).

Substituted: A fundamental compound, such as an aryl or aliphaticcompound, or a radical thereof, having coupled thereto, typically inplace of a hydrogen atom, another atom or group, i.e., a substituent.For example, substituted aryl compounds or substituents may have analiphatic group coupled to the closed ring of the aryl base, such aswith toluene. Again solely by way of example and without limitation, along-chain hydrocarbon may have a substituent bonded thereto, such asone or more halogens, oxygen such as a hydroxyl or ═O, an aryl group, acyclic group, a heteroaryl group or a heterocyclic group.

Target: A molecule for which the presence, location and/or concentrationis to be determined. Exemplary targets include proteins and nucleic acidsequences present in tissue samples.

Thiolate: A moiety having a formula —S—R, where R is an aryl, aliphaticor heteroaliphatic moiety.

II. Quinone Methide Analog Precursors

A. Overview

The present disclosure concerns compositions, kits and methods relatingto QMs and their precursors. QMPs of the present disclosure have beendeveloped for amplification of detection events using anenzyme-catalyzed conversion of quinone method precursors into reactivequinone methides, which can bind directly or proximally to the enzyme.

Quinone methides are quinone analogs where one of the carbonyl oxygenson the corresponding quinone is replaced by a methylene group (CH₂) toform an alkene, as shown below:

(See, e.g., Rokita, Quinone Methides, April 2009, John Wiley & Sons,Inc. which is hereby incorporated by reference herein for generaldisclosure related to quinone methides).

The methylene moiety of a quinone methide is an extremely reactiveelectrophile that will react with suitable reactive nucleophiles. Thereactive nucleophiles can be provided by a staining reagent enzyme, theantibody that the enzyme is conjugated to, and the biological sampleitself in immunohistochemistry applications. Generating quinone methidesin situ enables labels to be covalently bound to nucleophilic residuespresent within a matrix (e.g., tissue). Exemplary nucleophilic residuesinclude biological molecules comprising reactive nitrogen-, oxygen-, andsulfur-containing groups, such as amino, hydroxyl, and thiol groups ofamino acids (e.g., lysine, tyrosine, threonine, serine, and cysteine)and amino, carbonyl, and hydroxyl groups of nucleic acids.

Enzyme substrates capable of forming quinone methides (QMs) wereinitially investigated as potential mechanism-based inhibitors ofhydrolase enzymes. For example, QMPs were investigated for inhibitingsteroid sulfatase (STS), which catalyzes the desulfation of biologicallyinactive, sulfated steroids to biologically active steroids. Accordingto this approach, the QM generated by STS would react with the STS toinhibit its activity, for example, as a therapeutic approach (Ahmed etal. ChemBioChem. 2009; 10:1457; which is incorporated herein byreference for disclosure related to the use of QM for inhibitingenzymes).

According to another approach, Lenger et al. disclose profiling activesulfatases using quinone methide (QM) traps (i.e. activity-basedproteomic probes). To profile active sulfatases in health and disease,an activity-based proteomic tool directed against sulfatases, thequinone methide (QM) trap, was evaluated as an activity-based proteomicprobes (ABPPs). The QM trap concept applied by Lenger et al. involved insitu generation of a reactive QM intermediate that is dependent onenzymatic turnover of an enzyme inhibitor. Fluoromethylphenolate sulfatesubstrates were used as the QM precursor to generate a QM which wouldthen spontaneously fragment by fluoride elimination. The QM was used tocapture an active site residue conserved in the sulfatase, resulting inturnover-dependent inactivation and specific protein labelling. Thetraps were designed to have broad-ranged reactivity against sulfatases(Lenger et al. Bioorg Med Chem. Jan. 15, 2012; 20(2): 622-627, which ishereby incorporated by reference herein for disclosure related to theuse of QM traps for ABPP).

The approaches of Lenger et al. and Ahmed et al. are contrary to thepresently disclosed technology because enzyme inactivation is not a goalof the present embodiments. Instead, enzyme inactivation is to beavoided when the enzyme is being used to amplify detection usingQM-based labels. When used for detection, maintaining enzymatic activityis desirable so that each enzyme can produce greater signaling. Ingeneral, quenching the enzyme is contrary to the amplification objectivedescribed herein. In other terms, the QM precursor is selected and/ordesigned to avoid enzyme quenching. Instead, the QM precursor isselected and/or designed such that the QM can diffuse from the catalyticsite of the enzyme. While not inactivating the enzyme, the QM should besufficiently reactive with nucleophiles in the vicinity of the enzyme toprovide appropriate target labeling.

In yet another example, a disclosure by Qing Shao et al. described usinga QM precursor as a covalent reporter of beta-lactamase activity forfluorescent imaging and rapid screening of antibiotic-resistant bacteria(Shao, Q.; Zheng, Y.; Dong, X. M.; Tang, K.; Yan, X. M.; Xing, B. G.Chem-Eur J 2013, 19, 10903; which is hereby incorporated by referenceherein for disclosure related to the use of QM labels for fluorescentlylabeling whole bacterial cells). According to this approach, the QM isused as a fluorescent probe that can be activated by theresistance-associated beta-lactamase, which is a naturally occurringbacterial enzyme that destroys penicillin and cephalosporin antibiotics.The disclosed QM probe requires cleavage of a fluorescence-quenchingFluorescence Resonance Energy Transfer (FRET) group, along with theformation of a reactive quinone methide, which can then bind to theantibiotic-resistant bacteria. This approach relies on active endogenousenzymes and seeks to non-specifically label the entireantibiotic-resistant bacteria. Furthermore, the bacteria cells werestained in solution. This solution staining is advantageous because theconcentration of cells in solution dictates inter-cellular distance anddilution can be used to increase inter-cellular distances.

The present disclosure uses non-endogenous enzymes to avoid creatingfalse negatives in a staining protocol. QMPs disclosed herein weredesigned to bind proximally to the enzyme without inhibiting the enzyme(i.e. the QMPs are substantially non-inhibiting to the enzyme). Whilelong range diffusion of the reactive QM compound was irrelevantaccording to the approach of Qing Shao et al., the QM precursor of thepresent disclosure is selected and/or designed to be sufficientlyreactive to limit diffusion distances from the target. For example, whenused in a formalin-fixed paraffin embedded (FFPE) tissue sample, longrange diffusion of the reactive species would result in diffuse andblurred staining. Accordingly, overly stable reactive QM compounds wouldbe unsuitable for use in tissue staining. Examples included hereindemonstrate this unfavorable staining result when using overly stable QMcompound reactive compounds.

A recent publication from Kwan et al. (Angew Chem Int Edit 2011, 50,300, which is incorporated by reference herein for disclosure related tothe use of QM labels for fluorescently labeling) reported fluorescentplant histological staining utilizing coumarin glycosides modified togenerate QMs upon reaction with its cognate glycosidase. This reportdemonstrated the potential of QMs for covalent labeling of solid-phaseproteins with minimal diffusion from the site of generation, which isimperative for solid-phase immunoassays, and is an important feature ofthe current TSA technology. A key limitation of the probe described byKwan et al. is the requirement that each reporter molecule be modifiedsynthetically to contain QM precursor functionality. That is, inlabeling enzymes of interest in plant cells, the coumarin was modifiedto be both the quinone methide generating species and the label. Thisapproach adds significant cost and complexity to the generation ofvarious labels and would be unsuitable for many detectable labels (e.g.those detectable labels that have a structure which cannot be modifiedto include a quinone methide generating moiety). As such, Kwan et al.does not describe a QM detectable molecule with a separate quinonemethide generating moiety and a detectable moiety. Kwan et al. alsoestablish that the previously developed QM precursors have beenineffectively implemented. Accordingly, a need still persists in the artfor QM precursor compounds that comprise a separate QM generating moietyand a detectable moiety, and result in a QM that does not inhibit theenzyme. In particular, Kwan et al. concludes that the time required forthe (di)halomethyl phenol to decompose, and for the quinone methidethereby generated to react, is often sufficiently long that the reagentcan diffuse from the active site and react with other availablenucleophiles, including water. Further, Kwan et al. discloses QMprecursor compounds comprising a difluoromethyl moiety and describestheir superiority over the monofluoromethyl derivative based on greaterstability of the difluoromethyl QM precursor compound towards solvolysisand on greater stability of the fluoro-QM compound generated from theprecursor compound.

The currently described quinone precursors and methods of using the sameuse a generalized approach in which the detectable label and the quinonemethide generation and nucleophile stabilization functions are separatedwithin a molecule. One approach is to use a single QM precursor scaffoldcontaining an amine-functionalized linker group that allows simpleconjugation to nearly any detectable molecule. One important embodimentconcerns applying CARD to solid-phase immunoassays, such as IHC on FFPEtissue. Accordingly, using a phosphate group to exemplify theenzyme-cleavable recognition group was a logical choice due to theubiquity of its cognate enzyme alkaline phosphatase (AP) in currentimmunoassays.

Referring now to FIG. 1 , the application of AP-based CARD for IHCbegins with the incubation of a primary antibody (Ab) 102 with a sample.Ab 102 recognizes an antigen 104 of interest. The sample is thenincubated with a secondary Ab 106 that binds the primary Ab by typicalanti-species Ab binding. Secondary antibody 106 is labeled with anenzyme 108, for example alkaline phosphatase (AP). Thedetectable-labeled QM precursor 110 is then applied. Thedetectable-labeled QM precursor 110 includes a reporter group 112 and anenzyme recognition group 114 (a phosphate in this example). APrecognizes and cleaves the phosphate group, resulting in ejection of theleaving group, and the formation of a QM. These QMs either react withimmobilized tissue nucleophiles in close proximity to the site ofgeneration, or are quenched by nucleophiles in the reaction media. Thedetectable molecules that are covalently bound to the tissue are thendetected by one of a variety of visualization techniques in the case ofhaptens, or by fluorescence microscopy in the case of fluorophores.

B. Compounds

As disclosed herein, a QMP comprises a conjugated system that includesan enzyme recognition group, a leaving group, a detectable labelattached to the system through a linker, which may be a bond or may be alinker moiety. The conjugated system may be an aromatic system. Thesystem is conjugated such that when the enzyme recognition groupinteracts with the corresponding enzyme, and the leaving group leaves, aQM results.

The enzyme recognition group is selected on the basis of its suitabilityfor interaction with a particular enzyme. For example, for suitableinteraction with a phosphatase the enzyme recognition group is aphosphate (—P(O)(OH)₂), typically attached through an oxygen, nitrogenor sulfur to the conjugated system; for a phosphodiesterase, the enzymerecognition group is a phosphodiester; for an esterase, it is an ester;for an amidase or a protease, it is an amide; for a nitroreductase, itis a nitro group; for a urease, it is a urea group; for a sulfatase, itis a sulfate; for a cytochrome P450 enzyme, it is typically an alkoxy;for a lactamase, the enzyme recognition group is a β-lactam-containingmoiety; and for glucosidases, galactosidases and glucoronidases, it isan enzyme-appropriate sugar (e.g. alpha- or beta-glucose, alpha- orbeta-galactose, etc.) attached by an oxygen to the conjugated system.

The leaving group and the detectable label and linker may be part of thesame substituent of the conjugated system, and in some embodiments, theyare located adjacent, or ortho, to the enzyme recognition group.

In some embodiments, the staining amplification composition comprises aQMP according to formulas I and II

wherein A is a conjugated system such as a cyclic conjugated system withone or more rings, an acyclic conjugated system or a conjugated systemwith a combination of cyclic and acyclic features. In particularembodiments, conjugated system A is a substituted or unsubstituted arylring system, such as a carbocyclic aryl or heteroaryl ring system. ZR¹is an enzyme recognition group, or R¹ is an enzyme recognition group andZ is O, S or NR^(a), where R^(a) is hydrogen or aliphatic, typicallyalkyl and in some embodiments, lower alkyl. LG is a leaving group, andthe —C(LG)(R⁵)(R⁶) and —C(R⁵)LG- moieties are capable of forming analkene (C═C) functional group in a QM. Z- and LG-containing moieties arebound at relative positions on the conjugated system such that when R¹is cleaved from Z a transitional structure is formed that rearranges toeliminate LG to form a QM. Alternatively, —C(LG)(R⁵)(R⁶) and R¹ arepositioned ortho- to each other and together form a phosphodiester, withLG-ZR¹ being —O—P(O)(OH)O—. In such embodiments, a quinone methide isformed when the phosphodiester is cleaved from both Z and the—C(R⁵)(R⁶)— moiety.

Also with reference to formula I or II, R⁵ and R⁶ independently arehydrogen, halo, cyano, lower alkyl, lower haloalkyl, —C(O)alkyl,—C(S)alkyl, —C(O)OH, —C(O)Oalkyl, —C(O)NHR^(c) or —C(O)N(R^(c))₂ whereeach R^(c) independently is hydrogen, aryl, aliphatic orheteroaliphatic, or two R^(c) moieties together form a heteroaliphaticring. R³ is a linker or a bond, and R⁴ is a detectable label.

In some embodiments, LG is a halide, alkoxy, carboxylate, inorganicester, thiolate, amine, carboxylate or phenoxide. In other embodiments,LG is fluoro, chloro, azide, methoxy, ethoxy, isopropoxy, acetate,pyridium, DABCO (1,4-diazabicyclo[2.2.2]octane) or triethylamine. Insome embodiments, —C(R⁵)LG or —C(LG)R³— forms an epoxide ring where LGis the oxygen in the ring.

In some embodiments, the QMP has a formula III

where each Q independently is carbon or a heteroatom selected from O, Nor S and the ring has sufficient conjugation to allow the formation ofthe QM, such as an aryl ring or other conjugated system; each R⁷independently is ZR¹, a moiety comprising LG, a moiety comprising adetectable label, hydrogen, lone pair, halo, cyano, oxo (═O), aliphatic,alkoxy, NO₂, N(R^(c))₂, aryl, haloalkyl, —C(O)alkyl, —C(S)alkyl,—C(O)OH, —C(O)Oalkyl, —C(O)NHR^(c), —C(O)N(R^(c))₂, or two adjacent R⁷groups together form an aliphatic ring or aryl ring; m is 0 or 1; and Z,R¹, LG, R^(a) and R^(c) are as previously defined for formulas I and II.Also with reference to formula III, at least one R⁷ is ZR¹, at least oneR⁷ comprises LG, and the QMP comprises at least one detectable label. Insome embodiments, at least one R⁷ comprises a detectable label. In someembodiments, LG is the oxygen of an epoxide ring. A person of ordinaryskill in the art will appreciate that each R⁷ is also selected tosatisfy valence requirements. For example, if Q is oxygen, then R⁷ is alone pair.

In some embodiments, the conjugated ring is a 6-membered ring and ZR¹and the LG-containing moiety are ortho or para to each other.

In particular embodiments, the QMP has a formula IV

where Z is O, S or NR^(a) and R¹ is an enzyme recognition group, or ZR¹is an enzyme recognition group; R¹ is —C(LG)(R⁵)(R³R⁴), —R³R⁴ or—C(LG)(R⁵)(R⁶); R⁹, R¹¹ and R¹² are each independently hydrogen, halo,cyano, aliphatic, alkoxy, NO₂, N(R^(c))₂, aryl, haloalkyl, —C(O)alkyl,—C(S)alkyl, —C(O)OH, —C(O)Oalkyl, —C(O)NHR^(c), —C(O)N(R^(c))₂, —R³R⁴ ortwo adjacent groups together form an aliphatic ring or aryl ring; R¹⁰ ishydrogen, halo, cyano, aliphatic, alkoxy, NO₂, N(R^(c))₂, aryl,haloalkyl, —C(O)alkyl, —C(S)alkyl, —C(O)OH, —C(O)Oalkyl, —C(O)NHR^(c),—C(O)N(R^(c))₂, —R³R⁴, —C(LG)(R⁵)(R⁶) or with one of R⁹ or R¹¹ form analiphatic ring or aryl ring; each R^(a) independently is hydrogen oraliphatic, typically alkyl or lower alkyl; LG is a leaving group, or ZR¹and LG together form a phosphodiester; R³ is a bond or a linker; R⁴ is adetectable label; each R^(c) independently is hydrogen, aryl, aliphaticor heteroaliphatic, or two R^(c) moieties together form aheteroaliphatic ring; each R⁵ is independently hydrogen, halo, cyano,lower alkyl, lower haloalkyl, —C(O)alkyl, —C(S)alkyl, —C(O)OH,—C(O)Oalkyl, —C(O)NHR^(c) or —C(O)N(R^(c))₂; each R⁶ is independentlyhydrogen, halo, cyano, lower alkyl, lower haloalkyl, —C(O)alkyl,—C(S)alkyl, —C(O)OH, —C(O)Oalkyl, —C(O)NHR^(c) or —C(O)N(R^(c))₂; and ifLG is halo, then R⁵ and R⁶ are not halo. Also with reference to formulaIV, at least one of R⁸ and R¹⁰ comprises LG, and the QMP comprises atleast one —R³R⁴ moiety. In some embodiments, at least one of R⁸-R¹²comprises or consists of R³R⁴, and in certain embodiments, at least oneof R⁸ and R¹⁰ comprises or consists of R³R⁴.

Several exemplary analogs of formula IV include

where R¹³-R²⁰ are each independently hydrogen, halo, cyano, aliphatic,alkoxy, NO₂, N(R^(c))₂, aryl, haloalkyl, —C(O)alkyl, —C(S)alkyl,—C(O)OH, —C(O)Oalkyl, —C(O)NHR^(c), —C(O)N(R^(c))₂, —R³R⁴ or twoadjacent groups together form an aliphatic ring or aryl ring, and atleast one of R⁸-R²⁰ comprises or consists of R³R⁴.

Other particular exemplary analogs of formula IV include

LG may be a halide, alkoxy, carboxylate, inorganic ester, thiolate,amine, carboxylate or phenoxide. In other examples, LG is fluoro,chloro, azide, methoxy, ethoxy, isopropoxy, acetate, pyridium, DABCO(1,4-diazabicyclo[2.2.2]octane) or triethylamine. In certainembodiments, LG is F, Cl, —OS(O)₂CH₃, —OS(O)₂C₆H₄CH₃, —OS(O)₂C₆H₅,—OS(O)₂C₆H₄CX₃, —OC₆H₅, —N₂ ⁺, —NH₃ ⁺, —N₃, —NC₅H₅ ⁺, —O-alkyl—OC(O)alkyl, —OC(O)H, —N(R^(b))₃ ⁺ or DABCO, where X is F, Cl, Br or I,and each R^(b) independently is hydrogen or lower alkyl or two R^(b)moieties together form a heteroaliphatic ring.

In some embodiments, R³ is —(CH₂)_(n)NH—, —O(CH₂)_(n)NH—,—N(H)C(O)(CH₂)_(n)NH—, —C(O)N(H)(CH₂)_(n)NH—, —(CH₂)_(n)O—,—O(CH₂)_(n)O—, —O(CH₂CH₂O)_(n)—, —N(H)C(O)(CH₂)_(n)O—,—C(O)N(H)(CH₂)_(n)O—, —C(O)N(H)(CH₂CH₂O)_(n)—, —(CH₂)_(n)S—,—O(CH₂)_(n)S—, —N(H)C(O)(CH₂)_(n)S—, —C(O)N(H)(CH₂)_(n)S—,—(CH₂)_(n)NH—, —C(O)N(H)(CH₂CH₂O)_(n)CH₂CH₂NH—,—C(O)(CH₂CH₂O)_(n)CH₂CH₂NH—, —C(O)N(H)(CH₂)_(n)NHC(O)CH(CH₃)(CH₂)_(n)NH—or —N(H)(CH₂)_(n)NH—, where each n independently is 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11 or 12. In certain embodiments, R³ is —CH₂CH₂NH—,—OCH₂CH₂NH—, —NHCO(CH₂)₅NH—, —CONH(CH₂)₅NH—, —NHCO(CH₂)₆NH—,—CONH(CH₂)₆NH—, —CONH(CH₂)₂NH—, —(CH₂CH₂O)₄—, —(CH₂CH₂O)₈—,—C(O)N(H)(CH₂CH₂O)₂CH₂CH₂NH—, —CO(CH₂CH₂O)₄CH₂CH₂NH—,—CO(CH₂CH₂O)₈CH₂CH₂NH— or —C(O)N(H)(CH₂)₆NHC(O)CH(CH₃)(CH₂)₄NH—. R³ maycomprise a triazole, and in come embodiments, R³ is

In some embodiments, —C(LG)R³— or —C(LG)R⁵— forms an epoxide ring.

R⁴ may be a hapten, fluorophore, luminophore, or chromogen. In certainexamples, —R⁴ or linker-detectable label (—R³R⁴) is biotin conjugated tothe molecule by an aliphatic linker, nitropyrazole (NP), NP with a PEGlinker, such as a PEG-8 linker, TAMRA, DNP, Fast Red, HQ, HQ with a PEGlinker, such as a PEG-8 linker, benzofurazan, Rhod 110, Dabsyl with aPEG linker, such as a PEG-8 linker, or Cy.

In some embodiments, ZR¹ is —OP(O)(OH)₂, —SP(O)(OH)₂, —NR^(a)P(O)(OH)₂,—OC(═O)R^(a), —N(R^(a))C(═O)R^(a), —NO₂, —NR^(a)—C(═O)—N(R^(c))₂,—OSO₃H, —OR^(a), —O-β-lactam-containing moiety, —S-β-lactam-containingmoiety or —O-sugar where the sugar is an enzyme-appropriate sugar, suchas alpha- or beta-glucose, alpha- or beta-galactose, etc. or a saltthereof. In other embodiments, ZR¹ and LG together form aphosphodiester, —OP(O)(OH)O—.

In certain embodiments, LG is F, and in particular embodiments, LG is Fand R⁵ or R⁵ and R⁶ are H.

In certain embodiments of formula IV, R⁸ and ZR¹ together form aphosphodiester, leading to QMPs having a formula V

where R⁵, R⁶ and R⁹-R¹² are as previously defined for formula IV, and atleast one of R⁹—R¹² comprises or consists of R³R⁴. In particularembodiments, R¹⁰ comprises or consists of R³R⁴.

For certain exemplary embodiments of formula IV, the QMP is selectedfrom

In other exemplary embodiments of formula IV, the QMP is selected from

In the above examples, R⁴ is a detectable label, such as a hapten,fluorophore, luminophore, or chromogen. A person of ordinary skill inthe art will appreciate that the ZR¹, LG and R³ moieties shown in eachcase are exemplary moieties, and may be replace with any ZR¹, LG and R³moiety disclosed herein.

In some exemplary embodiments, the compound according to formula IVcomprises at least one detectable label and a moiety selected from

-   2-(2-((6-(amino)hexyl)amino)-1-fluoro-2-oxoethyl)phenyl dihydrogen    phosphate;-   2-(2-((2-aminoethyl)amino)-1-fluoro-2-oxoethyl)phenyl dihydrogen    phosphate;-   1-(2-((6-aminohexyl)amino)-1-fluoro-2-oxoethyl)naphthalen-2-yl    dihydrogen phosphate;-   2-(2-((4-(aminomethyl)benzyl)amino)-1-fluoro-2-oxoethyl)phenyl    dihydrogen phosphate;-   (2S,3S,4S,5R,6R)-6-(2-(2-((6-aminohexyl)amino)-1-fluoro-2-oxoethyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic    acid;-   N-(6-aminohexyl)-2-fluoro-2-(2-(((2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)phenyl)acetamide;-   (2S,3S,4S,5R,6S)-6-(2-(2-((6-aminohexyl)amino)-1-fluoro-2-oxoethyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic    acid;-   N-(6-aminohexyl)-2-fluoro-2-(2-(((2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)phenyl)acetamide;-   N-(6-aminohexyl)-2-fluoro-2-(2-(((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)phenyl)acetamide;-   (6R,7R)-7-acetamido-3-((2-(2-((6-aminohexyl)amino)-1-fluoro-2-oxoethyl)phenoxy)methyl)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic    acid;-   (6R,7R)-7-acetamido-3-(((2-(2-((6-aminohexyl)amino)-1-fluoro-2-oxoethyl)phenyl)thio)methyl)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic    acid;-   2-(2-((6-aminohexyl)amino)-1-fluoro-2-oxoethyl)phenyl hydrogen    sulfate;-   N-(6-aminohexyl)-2-fluoro-2-(2-methoxyphenyl)acetamide;-   2-(2-((6-aminohexyl)amino)-1-fluoro-2-oxoethyl)phenyl propionate;-   2-(2-((2-(2-(2-aminoethoxy)ethoxy)ethyl)amino)-1-fluoro-2-oxoethyl)phenyl    dihydrogen phosphate;-   2-(1-((6-aminohexyl)amino)-2-fluoro-1-oxopropan-2-yl)phenyl    dihydrogen phosphate;-   2-(2-acetamidophenyl)-N-(6-aminohexyl)-2-fluoroacetamide;-   N-(6-aminohexyl)-2-fluoro-2-(2-nitrophenyl)acetamide;-   N-(6-aminohexyl)-2-fluoro-2-(2-ureidophenyl)acetamide;-   N-(6-aminohexyl)-2-fluoro-2-(2-(((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)phenyl)acetamide;-   2-(2-((6-(2,6-diaminohexanamido)hexyl)amino)-1-fluoro-2-oxoethyl)phenyl    dihydrogen phosphate;-   2-((1-(2-aminoethyl)-1H-1,2,3-triazol-4-yl)fluoromethyl)phenyl    dihydrogen phosphate; or-   2-(fluoro(1H-1,2,3-triazol-4-yl)methyl)phenyl dihydrogen phosphate.

In other embodiments, the moiety is selected from

-   3-(2-((6-aminohexyl)amino)-1-fluoro-2-oxoethyl)-[1,1′-biphenyl]-4-yl    dihydrogen phosphate;-   2-(2-((6-aminohexyl)amino)-1-fluoro-2-oxoethyl)-4-(2-(4-hydroxyphenyl)acetamido)phenyl    dihydrogen phosphate;-   2-(2-((6-aminohexyl)amino)-1-fluoro-2-oxoethyl)-4-methoxyphenyl    dihydrogen phosphate;-   2-(2-((6-aminohexyl)amino)-1-fluoro-2-oxoethyl)-4-nitrophenyl    dihydrogen phosphate;-   4-acetamido-2-(2-((6-aminohexyl)amino)-1-fluoro-2-oxoethyl)phenyl    dihydrogen phosphate;-   4-(6-aminohexanamido)-2-(2-((6-aminohexyl)amino)-1-fluoro-2-oxoethyl)phenyl    dihydrogen phosphate;-   4-(6-aminohexanamido)-2-(fluoromethyl)phenyl dihydrogen phosphate;-   4-((6-aminohexyl)carbamoyl)-2-(fluoromethyl)phenyl dihydrogen    phosphate;-   4-(2-aminoethyl)-2-(fluoromethyl)phenyl dihydrogen phosphate;-   4-(6-aminohexanamido)-2-(1-fluoroethyl)phenyl dihydrogen phosphate;-   2-(6-aminohexanamido)-4-(1-fluoroethyl)phenyl dihydrogen phosphate;-   4-(6-aminohexanamido)-2-(2-(ethylamino)-1-fluoro-2-oxoethyl)phenyl    dihydrogen phosphate;-   4-(6-aminohexanamido)-2-(2-(butylamino)-1-fluoro-2-oxoethyl)phenyl    dihydrogen phosphate;-   6-amino-N-(2-hydroxy-2-oxido-4H-benzo[d][1,3,2]dioxaphosphinin-6-yl)hexanamide;-   2-(6-aminohexanamido)-4-(2-(ethylamino)-1-fluoro-2-oxoethyl)phenyl    dihydrogen phosphate;-   2-(6-aminohexanamido)-4-(2-(butylamino)-1-fluoro-2-oxoethyl)phenyl    dihydrogen phosphate; or-   2-((6-aminohexyl)carbamoyl)-4-(1-fluoroethyl)phenyl dihydrogen    phosphate;    where the detectable label is a hapten, fluorophore, luminophore, or    chromogen, and that the enzyme recognition group, leaving group and    linker moiety are exemplary, and may be replaced by any enzyme    recognition group, leaving group and linker moiety disclosed herein.

In other examples of formula II where the conjugated ring is phenyl, theQMP has a formula VI

wherein Z, LG, R¹, R³, R⁴, R⁵, R⁶, R^(a) and R^(c) are as previouslydefined for formula IV; R²¹, R²², R²³ and R²⁴ are each independentlyhydrogen, halo, cyano, aliphatic, alkoxy, NO₂, N(R^(c))₂, aryl,haloalkyl, —C(O)alkyl, —C(S)alkyl, —C(O)OH, —C(O)Oalkyl, —C(O)NHR^(c),—C(O)N(R^(c))₂, —R³R⁴, or two adjacent groups together form an aliphaticring or aryl ring.

In certain embodiments, LG is F, and in particular embodiments, LG is Fand R⁵ and R⁶, and/or R^(a), are hydrogen.

In some embodiments of formula VI, the QMP has a structure

In other embodiments of formula VI, the QMP has a structure selectedfrom

In other examples, the QMP has a structure selected from

In any of the above examples, R⁴ is a detectable label, such as ahapten, fluorophore, luminophore, or chromogen. A person of ordinaryskill in the art will appreciate that the ZR¹, LG and R³ moieties shownin each case are exemplary moieties, and may be replaced with any ZR¹,LG and R³ moiety disclosed herein.

In some exemplary embodiments, the compound according to formula VIcomprises at least one detectable label and a moiety selected from

-   4-(2-((6-aminohexyl)amino)-1-fluoro-2-oxoethyl)phenyl dihydrogen    phosphate;-   4-(2-((2-(2-(2-aminoethoxy)ethoxy)ethyl)amino)-1-fluoro-2-oxoethyl)phenyl    dihydrogen phosphate;-   4-(1-((6-aminohexyl)amino)-2-fluoro-1-oxopropan-2-yl)phenyl    dihydrogen phosphate;-   4-(2-((6-(2,6-diaminohexanamido)hexyl)amino)-1-fluoro-2-oxoethyl)phenyl    dihydrogen phosphate;-   N-(6-aminohexyl)-2-fluoro-2-(4-(((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)phenyl)acetamide;-   4-(2-((6-aminohexyl)amino)-1-fluoro-2-oxoethyl)phenyl propionate;-   (2S,3S,4S,5R,6R)-6-(4-(2-((6-aminohexyl)amino)-1-fluoro-2-oxoethyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic    acid;-   N-(6-aminohexyl)-2-fluoro-2-(4-(((2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)phenyl)acetamide;-   (2S,3S,4S,5R,6S)-6-(4-(2-((6-aminohexyl)amino)-1-fluoro-2-oxoethyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic    acid;-   (6R,7R)-7-acetamido-3-((4-(2-((6-aminohexyl)amino)-1-fluoro-2-oxoethyl)phenoxy)methyl)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic    acid;-   (6R,7R)-7-acetamido-3-(((4-(2-((6-aminohexyl)amino)-1-fluoro-2-oxoethyl)phenyl)thio)methyl)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic    acid;-   N-(6-aminohexyl)-2-fluoro-2-(4-(((2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)phenyl)acetamide;-   4-(2-((6-aminohexyl)amino)-1-fluoro-2-oxoethyl)phenyl hydrogen    sulfate;-   N-(6-aminohexyl)-2-fluoro-2-(4-methoxyphenyl)acetamide;-   N-(6-aminohexyl)-2-fluoro-2-(4-(((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)phenyl)acetamide;-   2-(4-acetamidophenyl)-N-(6-aminohexyl)-2-fluoroacetamide;-   N-(6-aminohexyl)-2-fluoro-2-(4-nitrophenyl)acetamide;-   N-(6-aminohexyl)-2-fluoro-2-(4-ureidophenyl)acetamide;-   4-(2-((6-aminohexyl)amino)-1-fluoro-2-oxoethyl)-2-methoxyphenyl    dihydrogen phosphate;-   4-(2-((6-aminohexyl)amino)-1-fluoro-2-oxoethyl)-2-nitrophenyl    dihydrogen phosphate;-   2-acetamido-4-(2-((6-aminohexyl)amino)-1-fluoro-2-oxoethyl)phenyl    dihydrogen phosphate;-   2-(6-aminohexanamido)-4-(2-((6-aminohexyl)amino)-1-fluoro-2-oxoethyl)phenyl    dihydrogen phosphate;-   4-(2-((6-aminohexyl)amino)-1-fluoro-2-oxoethyl)-2-(2-(4-hydroxyphenyl)acetamido)phenyl    dihydrogen phosphate; or-   4-(3-((6-aminohexyl)carbamoyl)oxiran-2-yl)-2,6-dimethoxyphenyl    dihydrogen phosphate;    where the detectable labels is a hapten, fluorophore, luminophore,    or chromogen, and that the enzyme recognition group, leaving group    and linker moiety are exemplary, and may be replaced by any enzyme    recognition group, leaving group and linker moiety disclosed herein.

FIG. 2(A) provides a table illustrating some additional exemplary QMprecursors.

In some embodiments, the compound is selected from

-   2-(fluoromethyl)-4-(6-(5-(2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)hexanamido)phenyl    dihydrogen phosphate;-   4-(6-(2-((2,4-dinitrophenyl)amino)acetamido)hexanamido)-2-(fluoromethyl)phenyl    dihydrogen phosphate;-   2-(fluoromethyl)-4-(1-(5-nitro-1H-pyrazol-3-yl)-1,29-dioxo-5,8,11,14,17,20,23,26-octaoxa-2,30-diazahexatriacontan-36-amido)phenyl    dihydrogen phosphate;-   (E)-4-(6-(4-((3-((4-chloro-2-methylphenyl)carbamoyl)-2-hydroxynaphthalen-1-yl)diazenyl)benzamido)hexanamido)-2-(fluoromethyl)phenyl    dihydrogen phosphate;-   4-(6-(3′,6′-bis(dimethylamino)-3-oxo-3H-spiro[isobenzofuran-1,9′-xanthene]-5-carboxamido)hexanamido)-2-(fluoromethyl)phenyl    dihydrogen phosphate;-   4-(6-(3′,6′-diamino-3-oxo-3H-spiro[isobenzofuran-1,9′-xanthene]-5-carboxamido)hexanamido)-2-(fluoromethyl)phenyl    dihydrogen phosphate;-   1-(6-((6-((3-(fluoromethyl)-4-(phosphonooxy)phenyl)amino)-6-oxohexyl)amino)-6-oxohexyl)-3,3-dimethyl-2-((1E,3E)-5-((Z)-1,3,3-trimethylindolin-2-ylidene)penta-1,3-dien-1-yl)-3H-indol-1-ium    chloride;-   2-(methoxymethyl)-4-(6-(5-(2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)hexanamido)phenyl    dihydrogen phosphate;-   5-(6-(5-(2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)hexanamido)-2-(phosphonooxy)benzyl    acetate;-   1-(5-(6-(5-(2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)hexanamido)-2-(phosphonooxy)benzyl)pyridin-1-ium;-   1-(5-(6-(5-(2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)hexanamido)-2-(phosphonooxy)benzyl)-1,4-diazabicyclo[2.2.2]octan-1-ium;-   N,N-diethyl-N-(5-(6-(5-(2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)hexanamido)-2-(phosphonooxy)benzyl)ethanaminium;-   5-(6-(5-(2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)hexanamido)-2-(phosphonooxy)benzyl    diethylcarbamate;-   2-(fluoromethyl)-4-((5-(5-(2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)pentyl)carbamoyl)phenyl    dihydrogen phosphate; or-   2-(fluoromethyl)-4-(2-(5-(2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)ethyl)phenyl    dihydrogen phosphate.

In other embodiments of formula II, the conjugated ring is a heteroarylor heterocyclic ring. Exemplary heteroaryl or heterocyclic ring include,but are not limited to, pyrazole, imidazole, pyridine, pyrimidine,pyridazine, pyrazine, pyrrole, furan, thiophene, indole, benzoxazole,benzimidazole, thiazole, oxazole, imidazole, or purine.

Some exemplary analogs of formula II where the conjugated ring is aheteroaryl or heterocyclic ring include:

where Z, R¹ and LG are as previously defined with respect to formula IV;R⁵ and R⁶ are each independently hydrogen, halo, cyano, lower alkyl,lower haloalkyl, —C(O)alkyl, —C(S)alkyl, —C(O)OH, —C(O)Oalkyl,—C(O)NHR^(c) or —C(O)N(R^(c))₂; R²⁵-R²⁹ are each independently hydrogen,halo, cyano, aliphatic, alkoxy, NO₂, N(R^(c))₂, aryl, haloalkyl,—C(O)alkyl, —C(S)alkyl, —C(O)OH, —C(O)Oalkyl, —C(O)NHR^(c),—C(O)N(R^(c))₂, —R³R⁴, or two adjacent groups together form an aliphaticring or aryl ring; each R^(c) independently is hydrogen, aryl, aliphaticor heteroaliphatic, or two R^(c) moieties together form aheteroaliphatic ring; and at least one of R⁵, R⁶ and R²⁵-R²⁹ comprisesor consists of R³R⁴. In some embodiments, when LG is halo, R⁵ and R⁶ arenot halo.

In some embodiments of formulas I-VI, when R¹ is —P(O)(OH)₂,—C(LG)(R⁵)(R⁶) is —CH₂F, —CH₂OH, —CH₂OCH₃, —CH₂OC(O)CH₃, —CH₂N(C₂H₅)₃ ⁺,—CH₂(—NC₅H₅)⁺, or —CH₂(DABCO)⁺ where DABCO is1,4-diazabicyclo[2.2.2]octane. In certain embodiments, —C(LG)(R⁵)(R⁶) is—CH₂F, —CH₂OCH₃, or —CH₂OCOCH₃.

i) Enzyme Recognition Group

The enzyme recognition group can be selected from any group that issuitable for enzyme recognition. In illustrative embodiments, the enzymerecognition group is selected from phosphate, phosphodiester, amide,nitro, urea, sulfate, methyl, ester, alpha- or beta-glucose,beta-lactam, alpha- or beta-galactose, alpha- or beta-lactose, andalpha- or beta-glucuronic acid. A person of ordinary skill in the artcan select an appropriate enzyme recognition group based on the enzymebeing used, such as, groups suitable for recognition by a phosphatase,phosphodiesterase, esterase, lipase, amidase, protease, nitroreductase,urease, sulfatase, cytochrome P450, alpha- or beta-glucosidase, alpha-or beta-lactamase, alpha- or beta-glucoronidase, alpha- orbeta-galactosidase, alpha- or beta lactase.

Embodiments provided below illustrate exemplary enzyme recognitiongroups for a particular QMP

Referring now to FIGS. 3(A)-(C), a sample 200 having a target 202 iscontacted by an antibody 204. Target 202 is shown being specificallybound by the antibody 204. Antibody 204 is conjugated to one or moreenzymes 206. When contacted with a QMP 208, enzyme 206 catalyzescleavage of an enzyme recognition group, exemplified by a phosphategroup, from QMP 208 to produce a phenol intermediate 210. The phenoleliminates the leaving group (LG) to produce a QM conjugate 212. QMconjugate 212 is a reactive electrophile capable of reacting withnucleophiles.

Referring now to FIG. 3(B), QM conjugate 212 can react with nucleophilespresent in sample 200, such as amine or sulfhydryl groups, asillustrated. A covalently bound complex 214 between the QM conjugate 212and the sample 200 forms when the electrophilic QM conjugate and thenucleophilic groups on the sample react. Since enzyme 206 was locatedproximally to target 202 through antibody 204, detectable label 216 iscovalently bound to sample 200 proximally to target 202. Thus, thedetectable label 216 can be detected to identify the presence of target202. This reaction occurs iteratively so that many complexes 214 areformed at each target, thereby amplifying the signal associated with thedetection event. The detectable label 216 can be any compound usefultherefore, such as a hapten, fluorophore, luminophore, or chromogen thatcan be detected by suitable means. The enzyme 206 may be bound directlyor indirectly to a target 202.

Referring now to FIG. 3(C), a person of ordinary skill in the art willunderstand that a competing reaction between QM conjugate 212 and asolvent (e.g. water) may result in the formation of non-bound compounds218. Non-bound compounds 218 may be washed away, thereby reducingindiscriminate staining of the sample at other locations.

Covalently bound compound 214 has a general structure according toformulas VII or VIII:

wherein A, Z and R³-R⁶ are as previously defined.

Alkaline phosphatase (ALP, ALKP) (EC 3.1.3.1) is a hydrolase enzymeresponsible for removing phosphate groups from many types of molecules,including nucleotides, proteins, and alkaloids. The process of removingthe phosphate group is called dephosphorylation. As the name suggests,alkaline phosphatases (also referred to as basic phosphatases) are mosteffective in an alkaline environment. Alkaline phosphatases have severalattributes that are advantageous relative to available enzymesincluding, for example: (1) alkaline phosphatase has a kcat/Kmapproximating the diffusion-controlled limit of 1×10⁹ liter/mole-sec;(2) alkaline phosphatase's optimal pH is 9-10, a pH suitable forsubsequent reaction of the QM; (3) alkaline phosphatases are very stableenzymes that resist thermal and chemical degradation better than mostenzymes; and (4) alkaline phosphatases are reasonably small and methodsof conjugation to other biological molecules have been developed.

Sulfatases (EC 3.1.6) are esterase enzymes that catalyze the hydrolysisof sulfate esters in many types of molecule including steroids,carbohydrates and proteins. They hydrolytically cleave sulfate estersthrough a unique catalytic aldehyde, which is introduced by apost-translational oxidation. Sulfatases are distributed in a wide rangeof tissues throughout the body.

Glycosidases, also known as glycoside hydrolases (EC 3.2.1), catalyzethe hydrolysis of glycosidic bonds in complex sugars. They are extremelycommon enzymes in nature and catalyze hydrolysis both O- andS-glycosides.

Lipases are enzymes that catalyze the hydrolysis of fats. They are asubclass of esterase enzymes, and are found in a wide range of organismsand tissue types.

β-lactamases (EC 3.5.2.6) are enzymes that open β-lactam rings by ahydrolysis mechanism. They are produced by some bacteria and can resultin resistance to β-lactam antibiotics, such as penicillins.

ii) Leaving Group

The leaving group can be any suitable group that can act as a leavinggroup to form a quinone methide when the QMP is contacted by a suitableenzyme. In some embodiments, the leaving group is a group that can leaveas an anion, with a formal negative charge. In other embodiments, theleaving group has a positive charge before leaving the QMP, and leavesas a neutral species. Suitable leaving groups include, but are notlimited to, halide, azide, sulfate ester, carboxylate, inorganic ester,thiolate, amine, aryloxy, alkoxy, or heteroaryl. In particularembodiments, LG is fluoride, chloride, acetate, methoxy, ethoxy,isopropoxy, phenoxide, —OS(O)₂CH₃, —OS(O)₂C₆H₄CH₃, —OS(O)₂C₆H₅,—OS(O)₂C₆H₄CX₃ where X is halo, —OC₆H₅, —N₂ ⁺, —NH₃ ⁺, —NC₅H₅ ⁺, —O—alkyl, —OC(O)alkyl, —OC(O)H, —N(R^(b))₃ ⁺ where each R^(b) independentlyis hydrogen or lower alkyl or two R^(b) moieties together form aheteroaliphatic ring, or DABCO.

iii) Detectable Labels

As shown in FIGS. 4 and 5 , QMPs are synthesized to allow the use ofmany different detectable labels or reporter moieties, such as haptens,dyes, and other detection tags (R⁴ in formulas I-VI), to determine thepresence of a target in a sample. Suitable detectable labels includeluminophores (phosphors, fluorophores), chromophores, and/or haptens. Aluminophore is a compound capable of luminescence, includingphosphorescence or fluorescence. Luminescence is the emission of lightby a compound caused by absorption of excitation energy in the form ofphotons, charged particles, or chemical changes. A fluorophore is afluorescent compound that absorbs light of a specific wavelength andre-emits light at a longer wavelength. A chromophore is a speciescapable of absorbing visible light. A preferred chromophore is capableof absorbing a sufficient quantity of visible light with sufficientwavelength specificity so that the chromophore can be visualized usingbright-field illumination. A hapten is a molecule, typically a smallmolecule that can combine specifically with an antibody, but typicallyis substantially incapable of being immunogenic except in combinationwith a carrier molecule. Certain luminophores, fluorophores, andchromophores also are haptens. Several exemplary detectable labels areshown in FIG. 2(A).

While not exhaustive, WO2012024185, which is incorporated in itsentirety herein by reference, provides disclosure concerning presentlyavailable chromogens and haptens. Embodiments of detectable labelsinclude haptens, such as pyrazoles, particularly nitropyrazoles;nitrophenyl compounds; benzofurazans; triterpenes; ureas and thioureas,particularly phenyl ureas, and even more particularly phenyl thioureas;rotenone and rotenone derivatives, also referred to herein as rotenoids;oxazole and thiazoles, particularly oxazole and thiazole sulfonamides;coumarin and coumarin derivatives; cyclolignans, exemplified byPodophyllotoxin and Podophyllotoxin derivatives; and combinationsthereof. Embodiments of haptens and methods for their preparation anduse are disclosed in U.S. Pat. No. 7,695,929, which is incorporated inits entirety herein by reference.

Exemplary haptens include, but are not limited to, BD (benzodiazepine),BF (benzofurazan), DABSYL (4-(dimethylamino)azobenzene-4′-sulfonamide,which has a λ_(max) of about 436 nm), DCC(7-(diethylamino)coumarin-3-carboxylic acid), DIG (digoxigenin), DNP(dinitrophenyl), HQ (3-hydroxy-2-quinoxalinecarbamide) NCA(nitrocinnamic acid), NP (nitropyrazole), PPT (Podophyllotoxin), Rhod(rhodamine), ROT (rotenone), and TS (thiazolesulfonamide). Othersuitable haptens include biotin and fluorescein derivatives (FITC(fluorescein isothiocyanate), TAMRA (tetramethylrhodamine), Texas Red).

Suitable chromophores include coumarin and coumarin derivatives.Exemplary coumarin-based chromophores include DCC and2,3,6,7-tetrahydro-11-oxo-1H,5H,11H-[1]benzopyrano[6,7,8-ij]quinolizine-10-carboxylicacid. Another class of chromogenic moieties suitable for use includesdiazo-containing chromogens, such as tartrazine, which has a λ_(max) ofabout 427 nm

In yet other embodiments, the chromophore may be a triarylmethanecompound. Exemplary triarylmethane chromophores are provided below:

Exemplary annulated chromophores include, but are not limited to:

and other rhodamine derivatives, such as tetramethylrhodamines(including TMR, TAMRA, and reactive isothiocyanate derivatives), anddiarylrhodamine derivatives, such as the QSY 7, QSY 9, and QSY 21 dyes.

Other exemplary detectable labels include resorufin; DAB; AEC; CN;BCIP/NBT; fast red; fast blue; fuchsin; NBT; ALK GOLD; Cascade Blueacetyl azide; Dapoxylsulfonic acid/carboxylic acid; DY-405; Alexa Fluor©405; Cascade Yellow; pyridyloxazole (PyMPO); Pacific Blue; DY-415;7-hydroxycoumarin-3-carboxylic acid; DYQ-425; 6-FAM phosphoramidite;Lucifer Yellow; iodoacetamide; Alexa Fluor© 430; Dabcyl; NBDchloride/fluoride; QSY 35; DY-485XL; Cy2; DY-490; Oregon Green 488carboxylic acid; Alexa Fluor© 488; BODIPY 493/503 C3; DY-480XL; BODIPYFL C3; BODIPY FL C5; BODIPY FL-X; DYQ-505; Oregon Green 514 carboxylicacid; DY-510XL; DY-481XL;6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein (JOE); DY-520XL;DY-521XL; BODIPY R6G C3; erythrosin isothiocyanate;5-carboxy-2′,4′,5′,7′-tetrabromosulfonefluorescein; Alexa Fluor© 532;6-carboxy-2′,4,4′,5′7,7′-hexachlorofluorescein (HEX); BODIPY 530/550 C3;DY-530; BODIPY TMR-X; DY-555; DYQ-1; DY-556; Cy3; DY-547; DY-549;DY-550; Alexa Fluor® 555; Alexa Fluor® 546; DY-548; BODIPY 558/568 C3;Rhodamine red-X; QSY 7; BODIPY 564/570 C3; BODIPY 576/589 C3;carboxy-X-rhodamine (ROX); Alexa Fluor® 568; DY-590; BODIPY 581/591 C3;DY-591; BODIPY TR-X; Alexa Fluor® 594; DY-594; carboxynaphthofluoresceinDY-605; DY-610; Alexa Fluor® 610; DY-615; BODIPY 630/650-X;erioglaucine; Alexa Fluor® 633; Alexa Fluor® 635; DY-634; DY-630;DY-631; DY-632; DY-633; DYQ-2; DY-636; BODIPY 650/665-X; DY-635; Cy5;Alexa Fluor® 647; DY-647; DY-648; DY-650; DY-654; DY-652; DY-649;DY-651; DYQ-660; DYQ-661; Alexa Fluor® 660; Cy5.5; DY-677; DY-675;DY-676; DY-678; Alexa Fluor® 680; DY-679; DY-680; DY-682; DY-681; DYQ-3;DYQ-700; Alexa Fluor® 700; DY-703; DY-701; DY-704; DY-700; DY-730;DY-731; DY-732; DY-734; DY-750; Cy7; DY-749; DYQ-4; and Cy7.5.

In some embodiments, the detectable label includes a linker moiety, suchas a PEG moiety. In certain embodiments, the addition of a PEG moietycan improve the staining intensity. FIGS. 6 and 7 illustrate theincrease in staining intensity of QMP-Dabsyl and QMP-Tamra derivativeswith PEG linkers, respectively. FIGS. 6(A)-6(C) provide microphotographsof Bcl-6 staining on tonsil tissue at 20× magnification, usingphosphate-QMP-Dabsyl (250 uM) (FIG. 6(A)), phosphate-QMP-PEG₄-Dabsyl(250 uM) (FIG. 6 (B)) and phosphate-QMP-PEG₈-Dabsyl (250 uM) (FIG.6(C)). As FIGS. 6(A)-6(C) show, the inclusion of a PEG linker in aphosphate-QMP-Dabsyl derivative increases the staining intensitysubstantially, compared to a phosphate-QMP-Dabsyl derivative without aPEG linker (FIGS. 6(A)-6(C)). Additionally, FIGS. 6(B) and 6(C)illustrate the difference in staining intensity between incorporatingPEG₄ (FIG. 6(B)) and PEG₈ (FIG. 6(C)) moieties into phosphate-QMP-Dabsylderivatives. Also, the PEG₈ moiety increases staining intensity relativeto the PEG₄ moiety.

A similar result is shown in FIGS. 7A-7 (C), which illustrate theincrease in staining intensity achieved by the incorporation of PEGlinkers into phosphate-QMP-Tamra derivatives. FIGS. 7A-7 (C) providemicrophotographs of Bcl-6 staining on tonsil tissue at 20×magnification, using phosphate-QMP-Tamra (250 uM) (FIG. 7(A)),phosphate-QMP-PEG₄-Tamra (250 uM) (FIG. 7 (B)) andphosphate-QMP-PEG₈-Tamra (250 uM) (FIG. 7(C)).

FIGS. 6 and 7 illustrates that incorporating PEG in the linker leads toan increase in functional staining intensity. However, it can also leadto an increase in diffusion of signal for the Tamra derivatives,especially with the PEG₈ linker (FIG. 7 (C)).

With reference to formulas I-VIII, in particular embodiments thedetectable label —R⁴ or linker-detectable label (—R³R⁴) is

In other particular embodiments, the detectable label —R⁴ orlinker-detectable label (—R³R⁴) is biotin with an aliphatic linker,nitropyrazole (NP), NP with a PEG-8 linker, TAMRA, DNP, Fast Red, HQ, HQwith a PEG-8 linker, benzofurazan, Rhod 110, Dabsyl with a PEG-8 linker,or Cy5. Quantum dots, lanthanide chelating polymers, and/or otherpolymer-based dyes and fluors may also be used.

iv) Linkers

Regarding the R³ linkers for formulas I-VIII, any suitable linker can beused to form conjugates of the present disclosure by coupling todetectable labels, such as chromogens, haptens, fluorophores, orluminophores, as disclosed herein. Useful linkers can either be homo- orheterobifunctional, but more typically are heterobifunctional.

Solely by way of example, and without limitation, a first class oflinkers includes aliphatic compounds, such as aliphatic hydrocarbonchains having one or more sites of unsaturation, or alkyl chains. Thealiphatic chain also typically includes terminal functional groups,including by way of example and without limitation, a carbonyl-reactivegroup, an amine-reactive group, a thiol-reactive group, carbon-reactivegroup or a photo-reactive group, that facilitate coupling to adetectable label as disclosed herein. The length of the chain can vary,but typically has an upper practical limit of about 30 atoms. Chainlinks greater than about 30 carbon atoms have proved to be lesseffective than compounds having smaller chain links. Thus, aliphaticchain linkers typically have a chain length of from about 1 carbon atomto about 30 carbon atoms. However, a person of ordinary skill in the artwill appreciate that, if a particular linker has greater than 30 atoms,and still operates efficiently for linking the detectable label to theQMP, and the conjugate still functions as desired, then such linkers arewithin the scope of the present disclosure.

A second class of linkers useful for practicing embodiments of thepresent disclosure is the alkylene oxides. The alkylene oxides arerepresented herein by reference to glycols, such as ethylene glycols. Aperson of ordinary skill in the art will appreciate that, as the numberof oxygen atoms increases, the hydrophilicity of the compound also mayincrease. Thus, suitable linkers may have a formula of (—OCH₂CH₂O—)_(n)where n is from about 2 to about 15, but more typically is from about 2to about 8.

Heterobifunctional polyalkyleneglycol linkers useful for practicingcertain disclosed embodiments are described in assignee's applications,including “Nanoparticle Conjugates,” U.S. patent application Ser. No.11/413,778, filed Apr. 28, 2006; “Antibody Conjugates,” U.S. applicationSer. No. 11/413,418, filed Apr. 27, 2006; and “Molecular Conjugate,”U.S. application Ser. No. 11/603,425, filed Nov. 21, 2006; all of whichapplications are incorporated herein by reference. Heterobifunctionalpolyalkyleneglycol linkers are disclosed below, and their useexemplified by reference to coupling tyramine to detectable labels.

One particular embodiment of a linker for use with disclosed conjugatesis a heterobifunctional polyalkyleneglycol linker having the generalstructure shown below:

wherein A and B include different reactive groups, x is an integer from2 to 10 (such as 2, 3 or 4), and y is an integer from 1 to 50, forexample, from 2 to 30 such as from 3 to 20 or from 4 to 12. One or morehydrogen atoms can be substituted for additional functional groups suchas hydroxyl groups, alkoxy groups (such as methoxy and ethoxy), halogenatoms (F, Cl, Br, I), sulfato groups and amino groups (including mono-and di-substituted amino groups such as dialkyl amino groups.

A and B of the linker independently are reactive functional groups, suchas a carbonyl-reactive group, an amine-reactive group, a thiol-reactivegroup, carbon-reactive group or a photo-reactive group. A and Btypically are not the same reactive functional group. Examples ofcarbonyl-reactive groups include aldehyde- and ketone-reactive groupslike hydrazine derivatives and amines. Examples of amine-reactive groupsinclude active esters such as NHS or sulfo-NHS, isothiocyanates,isocyanates, acyl azides, sulfonyl chlorides, aldehydes, glyoxals,epoxides, oxiranes, carbonates, aryl halides, imidoesters, anhydridesand the like. Examples of thiol-reactive groups includenon-polymerizable Michael acceptors, haloacetyl groups (such asiodoacetyl), alkyl halides, maleimides, aziridines, acryloyl groups,vinyl sulfones, benzoquinones, aromatic groups that can undergonucleophilic substitution such as fluorobenzene groups (such as tetraand pentafluorobenzene groups), and disulfide groups such as pyridyldisulfide groups and thiols activated with Ellman's reagent. Examples ofcarbon-reactive groups include halo-alkyl groups such as chloromethyl.Examples of photo-reactive groups include aryl azide and halogenatedaryl azides. Alternatively, A and/or B can be a functional group thatreacts with a specific type of reactive group. For example, A and/or Bcan be an amine group, a thiol group, or a carbonyl-containing groupthat will react with a corresponding reactive group (such as anamine-reactive group, thiol-reactive group or carbonyl-reactive group,respectively) that has been introduced or is otherwise present on ahapten and/or a tyramine or tyramine derivative. Additional examples ofeach of these types of groups will be apparent to those of ordinaryskill in the art. Further examples and information regarding reactionconditions and methods for exchanging one type of reactive group foranother are provided in Hermanson, “Bioconjugate Techniques,” AcademicPress, San Diego, 1996, which is incorporated by reference herein.

In some embodiments the heterobifunctional linker has the formula:

wherein A and B are different reactive groups and are as stated above; xand y are as stated above, and X and Y are additional spacer groups, forexample, spacer groups having between 1 and 10 carbons such as between 1and 6 carbons or between 1 and 4 carbons, and optionally containing oneor more amide linkages, ether linkages, ester linkages and the like.Spacers X and Y can be the same or different, and can bestraight-chained, branched or cyclic (for example, aliphatic or aromaticcyclic structures), and can be unsubstituted or substituted. Functionalgroups that can be substituents on a spacer include carbonyl groups,hydroxyl groups, halogen (F, Cl, Br and I) atoms, alkoxy groups (such asmethoxy and ethoxy), nitro groups, and sulfate groups.

In particular embodiments, the heterobifunctional linker comprises aheterobifunctional polyethylene glycol linker having the formula:

wherein n=1 to 50, for example, n=2 to 30 such as n=3 to 20 or n=4 to12. In particular embodiments, n=4 or 8.

Another class of linkers are aryl linkers. The aryl linkers can becarbocyclic or heterocyclic moieties, such as phenyl, pyridyl, pyrolyl,imidazolyl, pyrazolyl, triazolyl, oxazolyl or thiazolyl. In certainembodiments, the linker is a triazole, such as a 1,2,3 triazole. Thearyl group can be attached to the detectable label and/or QMP-moietiesthrough either a carbon or heteroatom. In an exemplary embodiment, thetriazole is formed by a reaction between an azide and an alkyne, such asa QMP moiety comprising an alkyne and an azide-functionalized detectablelabel, or a detectable label comprising an alkyne and anazide-functionalized QMP moiety (see, for example, Example 2, Scheme11). The azide and/or alkyne may be attached to the respective moietiesvia a linker group, such as a linker moiety disclosed herein, or may bedirectly attached through a covalent bond. In some embodiments, theazide and/or alkyne may be attached via an aliphatic chain, such as analkyl chain or lower alkyl chain. In other embodiments, the azide and/oralkyne is attached via a polyalkyleneglycol linker.

Several exemplary linkers are shown in FIGS. 2(A) and 2(B), third panel,and in the Examples.

III. Methods of Use

Embodiments of the disclosed QMs and their precursors are useful fordetecting targets, e.g., detecting a target in a biological sample.Detection can be performed, for example, using immunohistochemistrytechniques and/or in situ hybridization techniques.

In some embodiments, a target, such as a biomarker, within a tissue isdetected. In certain embodiments, the tissue is formalin-fixed,paraffin-embedded (FFPE) tissue. As understood by those skilled in theart, “tissue” as used herein may further comprise cervical cell smears,frozen tissues, circulating tumor cells on glass slides and bloodsmears. Thus, for example, the cervical cell smears may be used incytology preparations etc.

As now described with reference to FIG. 3(A)-(C) and detailed further inthe examples below, some method embodiments include contacting sample200 (e.g. tissue), which includes target 202 with antibody 204. In someembodiments, targets may be detected with a primary antibody that is notconjugated to enzymes, and a secondary antibody is used to detect theprimary antibody associated with the target. In either approach, theresult is localization of enzyme 206 in close proximity to target 202.The embodiment of FIG. 3 further includes contacting the tissue with aQMP conjugate 208 comprising (i) a phosphate or phosphodiester group and(ii) a detectable label or reporter 216. While shown as an antibody, anysuitable binding moieties may be used, for example nucleic acidoligomers, such as hapten labeled nucleic acid oligomers, and antibodiescapable of recognizing and binding to the target. Labeling the target202 with enzyme 206 may include contacting the tissue withenzyme-antibody conjugate comprising an antibody 204 to which the enzyme206 is linked. Antibody 204 is capable of recognizing and bindingspecifically to the target. The QMP 208 interacts with the enzyme 206 toform a phenol intermediate 210 that rearranges to form a QM 212, whichreacts with the binding moiety, the enzyme 206, the antibody 204, or thetissue to covalently link the detectable label 216 directly on orproximally to the target 202. The detectable label 216 then is detectedusing a method appropriate for the particular detectable label.

In particular embodiments, the QMP includes a phosphate orphosphodiester group, the enzyme is a phosphatase or phosphodiesterase,respectively, and the detectable label is a chromogen, a fluorophore, aluminophore, or a hapten. In some examples, alkaline phosphatase can beused as a phosphatase or a phosphodiesterase. In other embodiments, theQMP includes a β-galactoside, the enzyme is a β-galactosidase, and thedetectable label is a chromogen, a fluorophore, a luminophore, or ahapten.

In some embodiments, antibody 204 recognizes and binds directly totarget 202 as shown in FIG. 3(A). In other embodiments, an antibody maybe bound indirectly to any specific binding moiety. For example, ahapten-labeled, anti-binding moiety antibody may first be bound to thebinding moiety, followed by an anti-hapten antibody-enzyme conjugate.

Still further embodiments involve a method for forming animmunohistochemistry (IHC) or in situ hybridization (ISH) amplificationcomposition comprising a QMP. One exemplary embodiment comprises thestep of cleaving a phosphate or phosphodiester group covalently bound toa carbon or adjacent carbons, respectively, in a conjugated system(e.g., an aromatic ring system). When the QMP includes a phosphategroup, an electronic rearrangement results in elimination of a leavinggroup (LG) from a carbon ortho- or para- to the phosphate group. Thereaction is performed under conditions suitable for the formation of theQM. The precursor further comprises a detectable label bound to theconjugated system by a linker.

In some embodiments, the identity of the leaving group and/or the QMPconjugate concentration may influence target detection (e.g., staining)specificity. The reactivity of the QM depends at least in part on therate of QM formation, and therefore the leaving group ability of LG.Poor LGs may have a lower rate of QM formation, resulting in poorspecificity due to high QM stability and high diffusion from the site ofgeneration. In some embodiments, the identity of R⁵ and/or R⁶ alsoaffects target detection (e.g., staining) specificity. For example,certain groups (e.g., large groups) at R⁵ and/or R⁶ may stericallyhinder the QM's ability to react with and bind to a nucleophile.

In certain embodiments, when the leaving group was fluoride (e.g.,LG=F), superior results were obtained, resulting in specific, amplifiedIHC staining in which the stained areas when magnified have sharp,well-defined perimeters. Without being bound by a particular theory ofoperation, a fluoride leaving group may produce a more reactive QMP,resulting in rapid conversion to a QM and subsequent binding proximal tothe target. Less reactive QMPs may diffuse away from the target duringthe time between cleavage of the enzyme recognition group andelimination of the leaving group, followed by deposition and binding ofthe QM to a nucleophilic site. Additionally, nanomolar QMP conjugateconcentrations (e.g., 10 nM to 100 nM) may facilitate specific staining.

Further method embodiments relate to an immunohistochemistry or in situhybridization amplification method that includes contacting a samplewith an immunohistochemistry or in situ hybridization amplificationcomposition comprising a compound according to the structures disclosedherein; and contacting the amplification composition with a reagentunder conditions suitable to effect detection.

Yet other embodiments involve a method of detecting two or more distincttargets in a tissue sample. One exemplary embodiment comprises:contacting the tissue with a first binding moiety specific to a firsttarget, and a second binding moiety specific to a second target. Thefirst target is labeled with a first enzyme through the first bindingmoiety, and the second target is labeled with a second enzyme throughthe second binding moiety. The tissue is then contacted with a QMPcomprising (i) an enzyme recognition group, and (ii) a detectable label,and a second detection precursor compound (e.g., a second QMP or alabeled tyramide compound), wherein the QMP interacts with the firstenzyme to form a QM that reacts with the tissue to covalently link thedetectable label directly on or proximally to the first target, and thesecond detection precursor compound interacts with the second enzyme todeposit a second detection compound directly on or proximally to thesecond target. The detectable compounds are then detected.

Advantageously, for the methods just described, the first enzyme and thesecond enzyme are different enzymes. For example, the first enzyme canbe a phosphatase or phosphodiesterase, and the second enzyme can be aperoxidase. In certain embodiments, the first enzyme is alkalinephosphatase and the second enzyme is horseradish peroxidase. Alsoadvantageously, the first enzyme does not interact with the seconddetection precursor compound to deposit the second detection compoundproximally to the first target, and/or the second enzyme does notinteract with the QMP to form the QM. In other words, the first enzymereacts specifically with the QMP and the second enzyme reactsspecifically with the second detection precursor compound. For example,the first enzyme can be a phosphatase and the second enzyme aβ-galactosidase. In this example, the first enzyme, the phosphatase,does not react with the QMP comprising a β-galactoside, and the secondenzyme, the β-galactosidase, does not react with the QMP comprising thephosphate, Advantageously, these methods do not include an enzymedeactivation step.

In the interests of efficiency, the two or more target amplificationmethods may be practiced by contacting the tissue with the first bindingmoiety specific to the first target and contacting the tissue with thesecond binding moiety specific to the second target such that they occursubstantially contemporaneously. The first and second binding moietiesalso may be contacted with the first and second enzymes substantiallycontemporaneously. Additionally, the tissue may be contacted with theQMP and the second detection precursor compound substantiallycontemporaneously. However, a person of ordinary skill in the art willappreciate that the tissue may be serially contacted with the firstbinding moiety followed by the second binding moiety, and vice versa.Similarly, the first target may be labeled by the first enzyme followedby labeling the second target with the second enzyme, and vice versa.Also, the tissue may be serially contacted with the QMP followed by thesecond detection precursor compound, and vice versa.

FIGS. 8(A) and 8)B) and illustrate the results of simultaneous antibodyincubation followed by sequential chromogenic detection. In FIGS. 8(A)and 8(B), Her2 is detected by a GAR antibody conjugated to HRP, andPan-keratin is contacted with a GAM antibody conjugated to AP.Sequential detection by Tyramide-Tamra and QMP-PEG₈-Dabsyl resulted inthe image shown in FIG. 8(A). And FIG. 9 shows the results of aquadruplex sequential detection assay, detecting CD8 (Tyr-Rhodamine-110,maroon), CD3 (QMP-Cy5, blue), FoxP3 (Tyr-Tamra, purple) and Pan-keratin(QMP-Dabsyl, yellow) on tonsil tissue.

In certain embodiments, two targets are detected, either sequentially orsubstantially simultaneously, in a duplex assay. The detectionprecursors may be, for example, two QMPs comprising different enzymerecognition groups, or a QMP and a non-QMP-based detection method, suchas HRP-based TSA. In certain embodiments, a duplex assay comprises a QMPcomprising a phosphate, and a tyramine, for reaction with an alkalinephosphatase and HRP respectively. The detection method may bechromogenic or fluorescent IHC. FIG. 10 provides a microphotograph froma fluorescent duplex assay utilizing both AP-based QMP and HRP-based TSAdetections, but without an enzyme kill step. In other embodiments, aduplex assay comprises a QMP comprising a phosphate and a QMP comprisinga β-galactoside.

A triplex assay may be used to detect three targets. A triplex assay mayuse three QMPs with different enzyme recognition groups, or acombination of QMPs and other detection methods, such as HRP-based TSA.In certain embodiments, a triplex assay includes a QMP comprising aphosphate, a QMP comprising a β-galactoside and a tyramine, for reactionwith an alkaline phosphatase, a β-galactosidase and HRP respectively.The assay may be a sequential assay or a substantially simultaneousassay. In some embodiments, the detection method is chromogenic IHC.

A quadruplex assay may be used to detect four targets. A quadruplexassay may use four QMPs with different enzyme recognition groups, or acombination of QMPs and other detection methods, such as HRP-based TSA.In some embodiments, the quadruplex assay comprises the sequentialaddition of a QMP comprising a phosphate which reacts with an alkalinephosphatase, a QMP comprising a β-galactoside which reacts with aβ-galactosidase and two sequential additions of tyramine, each of whichreacts with HRP. In other embodiments, the quadruplex assay comprises aQMP comprising a phosphate which reacts with an alkaline phosphatase, aQMP comprising a β-galactoside which reacts with a β-galactosidase, andtyramine and DAB, each of which react with HRP. The detection method fora quadruplex may be chromogenic IHC.

In multiplexing assays that use different enzymes for each detectionstep, the presently described assays do not require enzyme kill steps,thereby reducing time and reagent waste. In addition, because the QM andTSA bind to different reactive sites on tissue, co-expressing markerscan be confidently detected without the possibility of the firstdetection step exhausting the reactive sites for the second.

Some embodiments concern a method for labeling an oligonucleotide. Anoligonucleotide is combined with a QMP comprising (i) a enzymerecognition group, and (ii) a detectable label as described herein. Anenzyme (e.g., a phosphatase, phosphodiesterase, etc.) then is added tothe combined oligonucleotide and QMP. The enzyme catalyzes conversion ofthe QMP into a reactive QM which covalently binds to the oligonucleotideto form a labeled oligonucleotide. In some embodiments, the detectablelabel is a hapten, such as a fluorophore. In one embodiment, the QMPcomprises a phosphate or phosphodiester group and the enzyme is alkalinephosphatase. In some embodiments, the QMP is used in an ISHamplification assay, such as to detect gene amplification.

In certain embodiments, the oligonucleotide is a detection probe capableof recognizing and binding specifically to a target within a sample,such as a biological sample. The method may further include contactingthe sample with the labeled oligonucleotide, whereby the labeledoligonucleotide binds to the target, and then detecting the target bydetecting the label. In contrast to some oligonucleotide labeling agents(e.g., aziridinium-based reagents (e.g., Mirus, Madison, WI)), certainembodiments of hapten- or fluorophore-labeled QMPs are not toxic.

Functional staining performance of QMPs is sensitive to several factorsincluding reaction time, temperature, substrate concentration, saltconcentration and the pH of the reaction media. An effective pH may befrom greater than 7 to 14, such as from 8 to 12, or from 9 to 11. TheQMP concentration may be from greater than zero to 1 mM or greater, suchas from 50 nM to 500 μM, from 50 nM to 100 μM, or from 100 nM to 1 μM.In certain embodiment concerning chromogenic staining, the concentrationis from 10 μM to 750 μM, or from 50 μM to 500 μM. In certain embodimentsconcerning fluorescent staining and/or hapten amplification, theconcentration is from 10 nM to 50 μM, or from 50 nM to 10 μM. Thesefactors allow both the intensity and diffusivity of the stain to bealtered in a predictable manner. However, a person of ordinary skill inthe art will appreciate that the staining conditions should also becompatible with the enzyme. For example, changing the buffer, pH,cofactors, etc. to such a degree that the enzyme's activity issubstantially reduced may have a negative effect on the stainingperformance of the QMP.

An example of the pH effect is illustrated by FIGS. 11 and 12 . Withoutbeing bound to a particular theory, at a relatively low pH of 7.5, fewerhydroxide nucleophiles were present in the reaction media. The resultwas longer QM lifetime, leading to more intense signal along withgreater diffusion (FIG. 11 ). Increasing the pH to 10 increased theconcentration of hydroxide nucleophiles, effectively decreasing QMlifetime. The result was decreased signal with much less diffusion (FIG.12 ).

An effective salt concentration may be from greater than zero to atleast 2 M, such from 0.1 M to 2 M, from 0.25 M to 1.5 M, or from 0.5 Mto 1.25 M. In certain embodiments, the salt concentration is about 1 M.The salt may be any salt effective to act as a cofactor of the enzyme,to improve signal intensity and/or to improve staining quality. Signalquality may be improved due to an improvement in signal localization,discreteness, and/or reduced diffusion. In some embodiments, the salt ismagnesium chloride or sodium chloride.

IV. Quinone Methide Analog Conjugates

Also within the scope of the present disclosure are conjugates includingembodiments of the disclosed QMs. In some embodiments, a conjugatecomprises a QM covalently bound to another substance, e.g., a biologicalsample, an oligonucleotide, an antibody, or an enzyme. The bound QM hasa general structure according to formulas VII or VIII:

wherein A is a conjugated system; Z is O, S or NR^(a) where R^(a) ishydrogen or aliphatic, typically alkyl; R³ is a bond or a linker; R⁴ isa detectable label; and R⁵ and R⁶ independently are hydrogen, halo,cyano, lower alkyl, lower haloalkyl, —C(O)alkyl, —C(S)alkyl, —C(O)OH,—C(O)Oalkyl, —C(O)NHR^(c) or —C(O)N(R^(c))₂ where each R^(c)independently is hydrogen, aryl, aliphatic or heteroaliphatic, or twoR^(c) moieties together form a heteroaliphatic ring. In certainexamples, R⁴ is a hapten, a chromogen, a fluorophore, or a luminophore.

V. Automated Embodiments

A person of ordinary skill in the art will appreciate that embodimentsof the method disclosed herein for using QMPs can be automated. VentanaMedical Systems, Inc. is the assignee of a number of United Statespatents disclosing systems and methods for performing automatedanalyses, including U.S. Pat. Nos. 5,650,327, 5,654,200, 6,296,809,6,352,861, 6,827,901 and 6,943,029, and U.S. published application Nos.20030211630 and 20040052685, each of which is incorporated herein byreference.

VI. Targets

Embodiments of the QMP and method disclosed herein may be used toidentify and/or quantify many different biological targets. Throughoutthis disclosure when reference is made to a target, it is understoodthat the target may be a target protein and that any polynucleotidesassociated with that protein can also be used as a target. The targetmay be a protein or nucleic acid molecule from a pathogen, such as avirus, bacteria, or intracellular parasite, such as from a viral genome.For example, a target protein may be produced from a targetpolynucleotide associated with (e.g., correlated with, causallyimplicated in, etc.) a disease. In certain disclosed embodiments, thetarget (or targets) of interest may be a particular nucleic acidsequence that may comprise a genetic aberration, such as a singlenucleotide polymorphism, promoter methylation, mRNA expression, siRNA, aparticular copy number change, a mutation, a certain expression level, arearrangement, or combination thereof. In some embodiments, the targetsare soluble proteins obtained from biological samples, such as serum,plasma, and/or urine. Some embodiments of the disclosed method may beused to detect and quantify DNA, RNA, and proteins of the same target(e.g., HER2) simultaneously from the same sample (e.g., from the sametissue section).

The disclosed method may be used to detect microRNA (miRNA or miR).MicroRNAs are small, non-coding RNAs that negatively regulate geneexpression, such as by translation repression. For example, miR-205regulates epithelial to mesenchymal transition (EMT), a process thatfacilitates tissue remodeling during embryonic development. However, EMTalso is an early step in tumor metastasis. Down-regulation of microRNAs,such as miR-205, may be an important step in tumor progression. Forinstance, expression of miR-205 is down-regulated or lost in some breastcancers. MiR-205 also can be used to stratify squamous cell andnon-small cell lung carcinomas (J. Clin. Oncol., 2009, 27(12):2030-7).Other microRNAs have been found to modulate angiogenic signalingcascades. Down-regulation of miR-126, for instance, may exacerbatecancer progression through angiogenesis and increased inflammation.Thus, microRNA expression levels may be indicative of a disease state.

A target nucleic acid sequence can vary substantially in size. Withoutlimitation, the nucleic acid sequence can have a variable number ofnucleic acid residues. For example a target nucleic acid sequence canhave at least about 10 nucleic acid residues, or at least about 20, 30,50, 100, 150, 500, 1000 residues. Similarly, a target polypeptide canvary substantially in size. The target polypeptides typically include atleast one epitope that binds to a peptide specific antibody, or fragmentthereof. In some embodiments that polypeptide can include at least twoepitopes that bind to a peptide specific antibody, or fragment thereof.

In specific, non-limiting examples, a target protein is produced by atarget nucleic acid sequence (e.g., genomic target nucleic acidsequence) associated with a neoplasm (for example, a cancer). Numerouschromosome abnormalities (including translocations and otherrearrangements, amplification or deletion) have been identified inneoplastic cells, especially in cancer cells, such as B cell and T cellleukemias, lymphomas, breast cancer, colon cancer, neurological cancersand the like. In some examples, therefore, at least a portion of thetarget molecule is produced by a nucleic acid sequence (e.g., genomictarget nucleic acid sequence) amplified or deleted in at least a subsetof cells in a sample. In one example, the genomic target nucleic acidsequence is selected to include a gene (e.g., an oncogene) that isreduplicated in one or more malignancies (e.g., a human malignancy).Oncogenes are known to be responsible for several human malignancies.For example, chromosomal rearrangements involving the SYT gene locatedin the breakpoint region of chromosome 18q11.2 are common among synovialsarcoma soft tissue tumors.

For example, HER2, also known as c-erbB2 or HER2/neu, is a gene thatplays a role in the regulation of cell growth (a representative humanHER2 genomic sequence is provided at GENBANK™ Accession No. NC_000017,nucleotides 35097919-35138441). The gene codes for a 185 kDatransmembrane cell surface receptor that is a member of the tyrosinekinase family. HER2 is amplified in human breast, ovarian, and othercancers; therefore, a HER2 gene (or a region of chromosome 17 thatincludes the HER2 gene) can be used as a genomic target nucleic acidsequence. Other breast cancer relevant proteins include the estrogenreceptor (ER) and progesterone receptor (PR).

In other examples, a target protein produced from a nucleic acidsequence (e.g., genomic target nucleic acid sequence) is selected thatis a tumor suppressor gene that is deleted (lost) in malignant cells.For example, the p16 region (including D9S1749, D9S1747, p16(INK4A),p14(ARF), D9S1748, p15(INK4B), and D9S1752) located on chromosome 9p21is deleted in certain bladder cancers. Chromosomal deletions involvingthe distal region of the short arm of chromosome 1 (that encompasses,for example, SHGC57243, TP73, EGFL3, ABL2, ANGPTL1, and SHGC-1322), andthe pericentromeric region (e.g., 19p13-19q13) of chromosome 19 (thatencompasses, for example, MAN2B1, ZNF443, ZNF44, CRX, GLTSCR2, andGLTSCR1) are characteristic molecular features of certain types of solidtumors of the central nervous system.

The aforementioned examples are provided solely for purpose ofillustration and are not intended to be limiting. Numerous othercytogenetic abnormalities that correlate with neoplastic transformationand/or growth are known to those of ordinary skill in the art. Targetproteins that are produced by nucleic acid sequences (e.g., genomictarget nucleic acid sequences), which have been correlated withneoplastic transformation and which are useful in the disclosed methods.

In other examples, a target protein is selected from a virus or othermicroorganism associated with a disease or condition. Detection of thevirus- or microorganism-derived target nucleic acid sequence (e.g.,genomic target nucleic acid sequence) in a cell or tissue sample isindicative of the presence of the organism. For example, the targetpeptide, polypeptide or protein can be selected from the genome of anoncogenic or pathogenic virus, a bacterium or an intracellular parasite(such as Plasmodium falciparum: and other Plasmodium species, Leishmania(sp.), Cryptosporidium parvum, Entamoeba histolytica, and Giardialamblia, as well as Toxoplasma, Eimeria, Theileria, and Babesiaspecies). In some examples, the target protein is produced from anucleic acid sequence (e.g., genomic target nucleic acid sequence) froma viral genome.

In certain examples, the target protein is produced from a nucleic acidsequence (e.g., genomic target nucleic acid sequence) from an oncogenicvirus, such as Epstein-Barr Virus (EBV) or a Human Papilloma Virus (HPV,e.g., HPV16, HPV18). In other examples, the target protein produced froma nucleic acid sequence (e.g., genomic target nucleic acid sequence) isfrom a pathogenic virus, such as a Respiratory Syncytial Virus, aHepatitis Virus (e.g., Hepatitis C Virus), a Coronavirus (e.g., SARSvirus), an Adenovirus, a Polyomavirus, a Cytomegalovirus (CMV), or aHerpes Simplex Virus (HSV).

VII. Kits

In illustrative embodiments, a kit includes a composition comprising aQMP disclosed herein. In certain embodiments, a kit also comprises oneor more enzyme-antibody conjugates, such as a phosphatase-antibodyconjugate or a phosphodiesterase-antibody conjugate. In some examples,the kit includes an alkaline phosphatase/anti-species antibody conjugateor an alkaline-phosphatase/anti-hapten antibody conjugate. The kit mayalso include a pH adjust, and/or enzyme cofactors.

In some embodiments, the QMP is stored in an organic solvent, such asDMSO, or in an organic solvent/aqueous buffer mixture, with up to 100%aqueous buffer. The aqueous buffer and/or the storage solution may havea pH of less than 7, such as from pH 0 to pH 5, or from pH 1 to pH 3,and in certain embodiments, the solution has a pH of about 2. Thestorage solution may also comprise one or more salts, such as magnesiumchloride or sodium chloride. In some embodiments, the concentration ofthe salt is from greater than zero to 2 M, such as from 0.25 M to about1.5 M, from 0.5 M to 1.25 M, or about 1 M. In exemplary embodiments, theQMP storage solution comprises the QMP in a mixture of DMSO and 10 mMglycine at pH 2, and with a concentration of magnesium chloride of up to1 M.

In some embodiments, the pH adjust is a solution suitable for adjustingthe pH of a solution from a pH suitable for storage of a QMP to a pHsuitable to enable effective use of a QMP in a staining and/oramplification assay. The storage pH may be from pH 0 to pH 5, and the pHsuitable for effective use of the QMP may be from pH 8 to pH 12. Incertain embodiments, the pH adjust comprises a Tris solution at about pH10, such as a 0.5 M Tris solution, or a Tris solution at about pH 8,such as a 0.25 mM Tris solution.

Exemplary options for the chromogen portion of the kit include, but arenot limited to:

-   -   3 dispensers—(i) QMP in 100% organic solvent; (ii) pH        adjust; (iii) enzyme cofactors;    -   3 dispensers—(i) QMP in organic/aqueous buffer mix (up to 100%        buffer); (ii) pH adjust; (iii) enzyme cofactors; and    -   2 dispensers—(i) QMP in organic/aqueous buffer mix (up to 100%        buffer)+enzyme cofactors; (ii) pH adjust.

A person of ordinary skill in the art will appreciate that in the kit,the enzyme and the antibody conjugates are typically stored separatelyto prevent unwanted reactions in storage.

VIII. Examples

The following examples are provided to illustrate certain specificfeatures of working embodiments and general protocols. The scope of thepresent invention is not limited to those features exemplified by thefollowing examples.

Example 1 Synthesis and Characterization of QMPs

Synthetic Materials and Methods. NMR data was collected on a Bruker 400MHz Spectrometer running Topspin (Bruker). Chemical shifts werereferenced to the deuterated solvent resonance for ¹H (7.26 ppm forCDCl₃, 2.50 ppm for DMSO-d₆, and 3.31 ppm for CD₃OD) and ¹³C (77.0 ppmfor CDCl₃, 39.51 ppm for DMSO-d6, and 49.15 ppm for CD₃OD). Chemicalshifts were referenced to external standards for ³¹P (0 ppm for H₃PO₄)and ¹⁹F (76.55 ppm for trifluoroacetic acid). MS data was collected on aJEOL ESI-TOF (AccuTOF JMS-T100LC) running Mass Center (JEOL). Prep HPLCwas performed on a Waters 2535 with Waters Sunfire columns (Prep C₁₈ OBD10 μm 50×250 mm) running Empower 3 (Waters). All chemicals werepurchased from commercial suppliers and used as received unlessotherwise noted.

Compound 2. 5-Nitrosalicylaldehyde (1) (10.0 g, 59.8 mmol) was suspendedin CH₂Cl₂ (100 mL) followed by addition of triethylamine (12.1 g, 120mmol) and diethyl chlorophosphate (15.5 g, 89.7 mmol) in a round bottomflask. The reaction mixture was stirred at room temperature for 1 hour.The reaction mixture was then extracted with 0.5M HCl (100 mL), theorganic layer collected and dried over MgSO₄. The suspension wasfiltered, the filtrate collected and the solvents removed under reducedpressure. The resulting residue was purified by flash chromatography intwo batches (hexanes:EtOAc) to give compound 2 as a colorless viscousoil (15.4 g, 85% yield): ¹H NMR (400 MHz, CDCl₃) δ 10.37 (s, 1H), 8.70(dd, J=8.4 Hz and 1.0 Hz, 1H), 8.42 (dd, J=9.0 Hz and 2.8 Hz, 1H), 7.69(dd, J=9.0 Hz and 1.0 Hz, 1H), 4.28 (m, 4H), 1.37 (m, 6H); ¹³C NMR (125MHz, CDCl₃) δ 186.2, 156.64, 156.58, 129.9, 127.5, 127.4, 124.5, 122.1,122.0, 65.7, 65.6, 16.1, 16.0; MS (ESI) m/z (M+H)⁺ calcd for C₁₁H₁₅NO₇P⁺304.1, found 303.7.

Compound 3. Compound 2 (3.50 g, 11.5 mmol) was dissolved in a mixture ofTHF and MeOH (1:1, 40 mL) followed by addition of NaBH₄ (655 mg, 17.3mmol) in a round bottom flask. The reaction mixture was stirred at roomtemperature for 2 hours. The reaction was then quenched with 0.5M HCl(40 mL) and the resulting solution extracted with EtOAc (3×100 mL). Theorganic layers were combined and dried over MgSO₄. The suspension wasfiltered, the filtrate collected and the solvents removed under reducedpressure. The resulting residue was purified by flash chromatography(hexanes EtOAc) to give compound 3 as a colorless viscous oil (2.55 g,73% yield): ¹H NMR (400 MHz, CDCl₃) δ 8.39 (d, J=2.4 Hz, 1H), 8.13 (dd,J=9.0 Hz and 2.8 Hz, 1H), 7.38 (dd, J=9.0 Hz and 1.0 Hz, 1H), 4.73 (s,2H), 4.24 (m, 4H), 1.37 (m, 6H); ¹³C NMR (125 MHz, CDCl₃) δ 152.6,152.5, 145.1, 134.6, 134.5, 125.3, 124.3, 121.1, 121.0, 65.65, 65.59,59.3, 16.12, 16.06; MS (ESI) m/z (M+H)⁺ calcd for C₁₁H₁₇NO₇P⁺ 306.1,found 305.7.

Compound 4. Compound 3 (2.00 g, 6.55 mmol) was dissolved in EtOAc (50mL) in a round bottom flask followed by addition of Pd/C (200 mg). Theflask was sealed and stirred under a H₂ atmosphere for 16 hours, atwhich point the reaction mixture was diluted with CH₂Cl₂ (10 mL). Aspatula-tip of celite was then added and the reaction mixture filtered.The filtrate was collected and the solvents removed under reducedpressure to give a colorless oil. The oil was dissolved in DMF (20 mL)followed by addition of DMAP (80 mg, 0.655 mmol), EDAC (1.38 g, 7.21mmol), and N-boc-aminocaproic acid (1.67 g, 7.21 mmol) in a round bottomflask. The reaction mixture was stirred at room temperature for 16hours, followed by quenching with H₂O (20 mL). The resulting emulsionwas extracted with EtOAc (3×100 mL), the organic layers combined anddried over MgSO₄. The suspension was filtered, the filtrate collectedand the solvents removed under reduced pressure. The resulting residuewas purified by flash chromatography (hexanes:EtOAc) to give compound 4as a colorless viscous oil (2.08 g, 65% yield): ¹H NMR (400 MHz, CDCl₃)δ 8.90 (s, 1H), 7.64 (dd, J=8.4 Hz and 2.4 Hz, 1H), 7.40 (d, J=2.4 Hz,1H), 7.04 (dd, J=9.0 Hz and 1.0 Hz, 1H), 4.84 (br s, 1H), 4.55 (s, 2H),4.41 (br s, 1H), 4.16 (m, 4H), 3.02 (t, J=6.8 Hz, 2H), 2.27 (t, J=7.2Hz, 2H), 1.63 (m, 2H), 1.44-1.40 (m, 11H), 1.38-1.32 (m, 8H); ¹³C NMR(125 MHz, CDCl₃) δ 172.0, 156.1, 143.6, 143.5, 136.25, 133.01, 132.96,121.3, 120.69, 120.67, 120.3, 79.0, 65.07, 65.01, 59.5, 40.3, 36.9,29.6, 28.3, 26.2, 25.1, 16.0, 15.9; MS (ESI) m/z (M+H)⁺ calcd forC₂₂H₃₇N₂NaO₈P⁺ 511.2, found 510.5.

Compound 5. Compound 4 (600 mg, 1.23 mmol) was dissolved in CH₂Cl₂ (10mL) in a sealed scintillation vial and cooled to 0° C. in an ice bath.Deoxo-Fluor® (bis(2-methoxyethyl)aminosulfur trifluoride, available fromSigma-Aldrich, 299 mg, 1.35 mmol) was then added drop wise and thereaction vessel sealed. The reaction mixture was stirred at 0° C. for 1hour, followed by quenching with H₂O (10 mL). The organic layer wasseparated and dried over MgSO₄. The suspension was filtered, thefiltrate collected and the solvents removed under reduced pressure. Theresulting residue was purified by prep RP-HPLC (0.05% TFA in H₂O:ACN) togive compound 5 as a colorless viscous oil (420 mg, 70% yield): ¹H NMR(400 MHz, CDCl₃) δ 8.69 (s, 1H), 7.64 (s, 1H), 7.44 (dd, J=9.0 and 0.8Hz, 1H), 7.19 (dd, J=9.0 Hz and 1.0 Hz, 1H), 5.38 (d, J=76 Hz, 2H), 4.71(br s, 1H), 4.18 (m, 4H), 3.06 (t, J=7.2 Hz, 2H), 2.29 (t, J=7.6 Hz,2H), 1.66 (m, 2H), 1.47-1.41 (m, 11H), 1.35-1.31 (m, 8H); ¹³C NMR (125MHz, CDCl₃) δ 171.8, 156.1, 144.06, 144.01, 143.99, 143.94, 135.8,127.74, 127.67, 127.57, 127.50, 121.3, 120.92, 120.85, 120.06, 80.5,78.9, 64.9, 64.8, 40.4, 36.9, 29.7, 28.3, 26.3, 25.0, 16.02, 15.95; MS(ESI) m/z (M+H)⁺ calcd for C₂₂H₃₇FN₂O₇P⁺ 491.2, found 491.5.

Compound 6. Compound 5 (140 mg, 0.286 mmol) was dissolved in CH₂Cl₂ (3mL) followed by addition of trimethylsilyl bromide (131 mg, 0.857 mmol)in a scintillation vial. The reaction vessel was sealed and the reactionmixture stirred at room temperature for 16 hours, at which point thereaction was quenched with MeOH (3 mL). The solvents were removed underreduced pressure and the resulting residue directly purified by prepRP-HPLC (0.05% TFA in H₂O:ACN) to give compound 6 as a white solid (61mg, 64% yield): ¹H NMR (400 MHz, CD₃OD) δ 7.68 (s, 1H), 7.52 (d, J=8.0Hz, 1H), 7.31 (d, J=8.0 Hz, 1H), 5.49 (d, J=48 Hz, 2H), 2.94 (t, J=8.0Hz, 2H), 2.42 (t, J=8.0 Hz, 2H), 1.78-1.66 (m, 4H), 1.51-1.43 (m, 2H);³¹P NMR (162 MHz, CD₃OD) δ −4.83; ¹⁹F NMR (376 MHz, CD₃OD) δ −77.5. MS(ESI) m/z (M+H)⁺ calcd for C₁₃H₂₁FN₂O₅P⁺ 335.1, found 334.7. ¹³C NMR wasnot determined due to low solubility and lack of signal.

Example of Conjugation to Compound 6:

Compound 7. Compound 6 (50 mg, 0.15 mmol) was dissolved in DMF (2 mL) ina scintillation vial, followed by addition of NHS-biotin (60 mg, 0.16mmol) and triethylamine (76 mg, 0.75 mmol). The reaction vessel wassealed and stirred at room temperature for 16 hours. The reactionmixture was directly purified by prep RP-HPLC 5 (0.05% TFA in H₂O:ACN)to give compound 7 as a white solid (46 mg, 58% yield): ¹H NMR (400 MHz,DMSO-d₆) δ 9.93 (s, 1H), 7.75 (t, J=5.7 Hz, 1H), 7.70 (s, 1H), 7.53 (d,J=8.8 Hz, 1H), 7.21 (d, J=8.8 Hz, 1H), 5.44 (d, J=47.6 Hz, 2H),4.36-4.24 (m, 1H), 4.17-4.06 (m, 1H), 3.09 (q, J=6.0 Hz, 1H), 3.02 (q,J=6.5 Hz, 2H), 2.81 (dd, J=12.4, 5.0 Hz, 1H), 2.57 (d, J=12.4 Hz, 1H),2.28 (t, J=7.4 Hz, 2H), 2.03 (t, J=7.3 Hz, 2H), 1.67-1.18 (m, 12H). ¹³CNMR (101 MHz, DMSO-d₆) δ 171.8, 171.1, 162.7, 135.6, 120.7, 120.4,119.7, 119.6, 80.5, 78.9, 61.0, 59.2, 55.4, 38.3, 36.2, 35.2, 29.0,28.2, 28.0, 26.1, 25.3, 24.8. ³¹P NMR (162 MHz, DMSO-d₆) δ −5.21; ¹⁹FNMR (376 MHz, DMSO-d₆) δ −76.57. MS (ESI) m/z (M−H)⁻ calcd forC₂₃H₃₃FN₄O₇PS⁻ 559.2, found 559.0.

Compound 9. N-Boc-tyramine (10.0 g, 42.1 mmol) 8 was dissolved in CHCl₃(80 mL) followed by addition of H₂O (40 mL) in a round bottom flaskequipped with a reflux condenser. Powdered NaOH (16.8 g, 421 mmol) wasthen added and the reaction mixture heated to 60° C. in an oil bath withvigorous stirring. After 1 hour, a second portion of NaOH (8.4 g, 210mmol) was added and the stirring continued for an additional 1 hour.After 1 hour, a third portion of NaOH (8.4 g, 210 mmol) was added andthe stirring continued for an additional 5 hours. The reaction mixturewas then extracted with 0.5M HCl, the organic layer collected and driedover MgSO₄. The suspension was filtered, the filtrate collected and thesolvents removed under reduced pressure. The resulting residue waspurified by flash chromatography (hexanes:EtOAc) to give compound 9 asan off-white low-melting solid (5.43 g, 49% yield): ¹H NMR (400 MHz,CDCl₃) δ 10.9 (s, 1H), 9.86 (s, 1H), 7.36 (d, J=20 Hz, 2H), 6.94 (d,J=20 Hz, 1H), 4.58 (br s, 1H), 3.34 (br s, 2H), 2.79 (t, J=6.8 Hz, 2H),1.43 (s, 9H); ¹³C NMR (125 MHz, CDCl₃) δ 196.5, 160.3, 155.9, 137.6,133.4, 130.4, 120.5, 117.8, 79.4, 41.7, 35.1, 28.4; MS (ESI) m/z (M−H)⁻calcd for C₁₄H₁₈NO₄ ⁻ 264.1, found 264.1.

Compound 10. Compound 9 (2.00 g, 7.54 mmol) was dissolved in CH₂Cl₂ (50mL) followed by addition of triethylamine (1.53 g, 15.1 mmol) anddiethyl chlorophosphate (1.95 g, 11.3 mmol) in a round bottom flask. Thereaction mixture was stirred at room temperature for 4 hours. Thereaction mixture was then extracted with 0.5M HCl (50 mL), the organiclayer collected and dried over MgSO₄. The suspension was filtered, thefiltrate collected and the solvents removed under reduced pressure. Theresulting residue was purified by flash chromatography (hexanes:EtOAc)to give compound 10 as a colorless viscous oil (2.20 g, 73% yield): ¹HNMR (400 MHz, CDCl₃) δ 10.3 (s, 1H), 7.66 (s, 1H), 7.38-7.34 (m, 2H),4.66 (br s, 1H), 4.21 (m, 4H), 3.32 (br s, 2H), 2.78 (t, J=7.2 Hz, 2H),1.38-1.30 (m, 15H); ¹³C NMR (125 MHz, CDCl₃) δ 188.4, 155.7, 151.4,151.3, 136.5, 136.0, 128.6, 127.10, 127.04, 121.17, 121.14, 79.2, 65.03,64.97, 41.4, 35.3, 28.3, 16.02, 15.95; MS (ESI) m/z (2M+Na)⁺ calcd forC₃₆H₅₆N₂NaO₁₄P₂ ⁺ 825.3, found 825.3.

Compound 11. Compound 10 (1.00 g, 2.49 mmol) was dissolved in a mixtureof THF and MeOH (1:1, 10 mL) followed by addition of NaBH₄ (141 mg, 3.74mmol) in a round bottom flask. The reaction mixture was stirred at roomtemperature for 3 hours. The reaction was then quenched with 0.5M HCl(10 mL) and the resulting solution extracted with CH₂Cl₂ (3×25 mL). Theorganic layers were combined and dried over MgSO₄. The suspension wasfiltered, the filtrate collected and the solvents removed under reducedpressure. The resulting residue was purified by flash chromatography(hexanes:EtOAc) to give compound 11 as a colorless viscous oil (540 mg,53% yield): ¹H NMR (400 MHz, CDCl₃) δ 7.29 (s, 1H), 7.14 (s, 2H), 4.65(s, 2H), 4.60 (br s, 1H), 4.27 (m, 4H), 3.37 (br s, 2H), 2.80 (t, J=6.8Hz, 2H), 1.45 (s, 9H), 1.39 (t, J=6.8 Hz, 6H); ¹³C NMR (125 MHz, CDCl₃)δ 155.8, 146.9, 146.8, 136.8, 133.04, 133.00, 131.4, 129.5, 121.12,121.10, 79.3, 65.13, 65.07, 60.2, 41.6, 35.5, 28.4, 16.11, 16.04; MS(ESI) m/z (M+H)⁺ calcd for C₁₈H₃₁NO₇P⁺ 404.2, found 404.2.

Compound 12. Compound 11 (250 mg, 0.620 mmol) was dissolved in CH₂Cl₂ (5mL) in a sealed scintillation vial and cooled to 0° C. in an ice bath.Deoxo-Fluor® (bis(2-methoxyethyl)aminosulfur trifluoride, available fromSigma-Aldrich, 151 mg, 0.682 mmol) was then added drop wise and thereaction vessel sealed. The reaction mixture was stirred at 0° C. for 15minutes, followed by quenching with H₂O (5 mL). The organic layer wasseparated and dried over MgSO₄. The suspension was filtered, thefiltrate collected and the solvents removed under reduced pressure. Theresulting residue was purified by prep RP-HPLC (0.05% TFA in H₂O:ACN) togive compound 12 as a colorless viscous oil (110 mg, 44% yield): ¹H NMR(400 MHz, CDCl₃) δ 7.30-7.25 (m, 2H), 7.15 (d, J=8.4 Hz, 1H), 5.45 (d,J=48 Hz, 2H), 4.70 (br s, 1H), 4.25-4.17 (m, 4H), 3.34 (br s, 2H), 2.78(t, J=7.2 Hz, 2H), 1.42 (s, 9H), 1.34 (t, J=6.8 Hz, 6H); ¹³C NMR (125MHz, CDCl₃) δ 155.8, 146.91, 146.86, 146.84, 146.79, 136.0, 130.3,129.7, 129.6, 127.6, 127.5, 127.4, 127.3, 119.9, 80.5, 79.2, 78.8,64.82, 64.76, 41.6, 35.3, 28.2, 16.0, 15.9; MS (ESI) m/z (M+H)⁺ calcdfor C₁₈H₃₀FNO₆P⁺ 406.2, found 405.7.

Compound 13. Compound 12 (110 mg, 0.247 mmol) was dissolved in CH₂Cl₂ (1mL) followed by addition of trimethylsilyl bromide (TMSB) (113 mg, 0.740mmol) in a scintillation vial. The reaction vessel was sealed and thereaction mixture stirred at room temperature for 16 hours, at whichpoint the reaction was quenched with MeOH (1 mL). The solvents wereremoved under reduced pressure and the resulting residue directlypurified by prep RP-HPLC (0.05% TFA in H₂O:ACN) to give compound 13 as awhite solid (45 mg, 72% yield): ¹H NMR (400 MHz, DMSO-d6) δ 7.25 (d,J=9.6 Hz, 2H), 7.17 (d, J=8.4 Hz, 1H), 5.38 (d, J=48 Hz, 2H), 3.00 (t,J=7.6 Hz, 2H), 2.83 (t, J=7.6 Hz, 2H); ³¹P NMR (162 MHz, DMSO-d6) δ0.22; ¹⁹F NMR (376 MHz, DMSO-d₆) δ −75.6. MS (ESI) m/z (M−H)⁻ calcd forC₉H₁₂FNO₄P⁻ 248.0, found 248.0. ¹³C NMR was not determined due to lowsolubility and lack of signal.

Example of Detectable Label Conjugation to Compound 13:

Compound 14. Compound 13 (15 mg, 0.060 mmol) was dissolved in DMF (2 mL)in a scintillation vial, followed by addition of NHS-biotin (23 mg,0.066 mmol) and triethylamine (18 mg, 0.18 mmol). The reaction vesselwas sealed and stirred at room temperature for 16 hours. The reactionmixture was directly purified by prep RP-HPLC (0.05% TFA in H₂O:ACN) togive compound 14 as a white solid (25 mg, 86% yield): ¹H NMR (400 MHz,CD₃OD) δ 7.33-7.21 (m, 3H), 5.42 (d, J=48 Hz, 2H), 4.52-4.49 (m, 1H),4.31-4.28 (m, 1H), 3.43-3.40 (m, 2H), 3.19-3.17 (m, 1H), 2.96-2.92 (m,1H), 2.83-2.79 (m, 2H), 2.72 (d, J=13 Hz, 1H), 2.15 (t, J=7.2 Hz, 2H),1.71-1.57 (m, 4H), 1.48-1.38 (m, 2H); ¹³C NMR (125 MHz, CD₃OD) δ 176.3,166.3, 149.15, 149.09, 149.04, 137.4, 131.4, 130.9, 130.8, 129.7, 129.6,129.5, 129.4, 121.5, 81.8, 80.1, 63.6, 61.8, 57.1, 41.8, 41.2, 36.9,35.8, 29.8, 29.6, 27.1; ³¹P NMR (162 MHz, CD₃OD) δ −5.12; ¹⁹F NMR (376MHz, CD₃OD) δ −77.3. MS (ESI) m/z (M−H)⁻ calcd for C₁₉H₂₆FN₃O₆PS⁻ 474.1,found 474.0.

Additional examples of detectable label conjugation to compound 13 areshown in Scheme 3.

Additional QMPs can be synthesized according to Schemes 4-10.

Compound 15 is treated with sodium borohydride in THF and methanol toform compound 16. Compound 16 is then treated first withN-hydroxysuccinimide in the presence of a carbodiimide crosslinkingagent such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC), inDMF to form an activated acid (not shown). The activated acid compoundis then reacted with 1-BOC pentanediamine in DMF and in the presence ofa base such as triethylamine, to form compound 17. Compound 17 is thenreacted with diethyl chlorophosphate and triethylamine indichloromethane to form compound 18, by the method used to make compound10 in Scheme 2, above. Compound 18 is then reacted with Deoxo-Fluor® indichloromethane to form compound 19, which in turn, is reacted withtrimethylsilyl bromide (TMSB) to form the deprotected amine compound 20.Compound 20 is then conjugated to biotin to form compound 21. Themethods to make compounds 19, 20 and 21 are the same as those used forcompounds 12, 13 and 14 in Scheme 2, above.

Compound 23. Phenol (22) (75.3 g, 800 mmol), glyoxylic acid monohydrate(9.21 g, 100 mmol), tributylamine (17.6 g, 22.6 mL, 95 mmol), Al₂(SO₄)₃(3.33 g, 5 mmol), and H₂O (4 mL) were combined in a round bottom flaskand the reaction mixture was heated to 50° C. for 8 hours. The reactionmixture was cooled to room temperature followed by addition of 1M NaOH(100 mL) and extraction with 1,2-dichloroethane (3×100 mL). The NaOHlayer was then acidified to pH=2 by careful addition of conc. HCl. Theresulting solution was extracted with EtOAc (5×200 mL). The organicswere collected, combined and dried over MgSO₄. The solvents were thenremoved under reduced pressure, giving compound 23 as a viscous lightbrown oil (13.4 g, 80% yield).

Compound 24. Compound 23 (12.0 g, 71.4 mmol) and N-boc-diaminohexane(23.2 g, 107 mmol) were dissolved in DMF (125 mL) followed by sequentialaddition of HOBt (965 mg, 7.14 mmol), EDAC (20.5 g, 107 mmol), andfinally DIEA (13.8 g, 18.6 mL, 107 mmol). The reaction mixture wasstirred under N₂ for 16 hours, followed by reduction of the DMF to about25 mL under reduced pressure. The resulting mixture was then quenchedwith 1M HCl (100 mL) and extracted with EtOAc (3×100 mL). The organicswere collected, combined, and dried over MgSO₄. The solvent was removedunder reduced pressure and the resulting residue was purified by flashchromatography in three separate and equal portions (Biotage Snap 50;hex:EA 1:0 to 5:95) to give compound 24 as a colorless viscous oil (16.0g, 61% yield). MS (ESI) m/z (M+2H-boc)⁺ calcd for C₁₄H₂₃N₂O₃ ⁺ 267.2,found 266.6.

Compound 25. Compound 24 (13.0 g, 35.5 mmol) was dissolved in EtOAc (25mL) and cooled to 0° C. in an ice bath under N₂. Triethylamine (10.8 g,14.8 mL, 107 mmol) was then added and allowed to stir for 10 minutes.Diethyl chlorophosphate (6.73 g, 7.69 mL, 39.0 mmol) was then addeddropwise over a period of 5 minutes. The reaction mixture was removedfrom the ice bath and stirred under N₂ for 4 hours. The reaction mixturewas then quenched with 1M HCl (200 mL) and extracted with EtOAc (3×200mL). The organics were collected, combined, and dried over MgSO₄. Thesolvent was removed under reduced pressure and the resulting residue waspurified by flash chromatography (Biotage Snap 340; hex:EA 1:0 to 5:95)to give compound 25 as a colorless viscous oil (16.2 g, 91% yield). MS(ESI) m/z (M+H)⁺ calcd for C₂₃H₄₀N₂O₈P⁺ 503.5, found 503.2.

Compound 26. Compound 25 (13.0 g, 25.9 mmol) was dissolved in dry CH₂Cl₂(100 mL) followed by cooling to −40° C. in an ice bath under N₂.Deoxo-Fluor® (6.02 g, 5.01 mL, 27.2 mmol) was then added drop wise overa period of 15 minutes. The reaction mixture was removed from the icebath and stirred under N₂ for 1 hour. The reaction mixture was thenquenched with a saturated NaHCO₃ solution followed by extraction withCH₂Cl₂ (3×100 mL). The organics were collected, combined, and dried overMgSO₄. The solvent was removed under reduced pressure and the resultingresidue was purified by flash chromatography (Biotage Snap 340;CH₂Cl₂:MeOH 1:0 to 92:8) to give compound 26 as a light yellow viscousoil (11.5 g, 85% yield). MS (ESI) m/z (M+2H-boc)⁺ calcd forC₁₈H₃₁FN₂O₅P⁺ 405.2, found 405.0.

Compound 27. Compound 26 (11.0 g, 21.8 mmol) was dissolved in dry CHCl₃(20 mL) followed by cooling to 0° C. in an ice bath under N₂. TMSBr(16.7 g, 14.3 mL, 109 mmol) was then added drop wise over a period of 10minutes. The reaction mixture was then removed from the ice bath andstirred under N₂ for 16 hours. The reaction mixture was quenched withMeOH (50 mL) and solvents were removed under reduced pressure. Theresidue was purified in five equal portions using prep RP-HPLC (C₁₈,50×250 mm, 40 mL/minute, 0.05% TFA in H₂O:CH₃CN 99:1 to 5:95 over 40minutes) to give compound 27 as a white solid (4.20 g, 55% yield). MS(ESI) m/z (M+H)⁺ calcd for C₁₄H₂₂FN₂O₅P⁺ 349.3, found 348.9.

Example of Conjugation to Compound 27: Compound 28.5(6)-Carboxytetramethylrhodamine (500 mg, 1.16 mmol) was dissolved indry DMSO (5 mL) followed by addition of DMAP (213 mg, 1.74 mmol) andN,N′-disuccinimidyl carbonate (327 mg, 1.28 mmol). The reaction vesselwas sealed and the reaction mixture stirred at room temperature for 30minutes. Compound 27 (446 mg, 1.28 mmol) was then added, followed byaddition of DIEA (750 mg, 1.01 mL, 5.80 mmol). The resulting mixture wasstirred at room temperature for 2 hours. The reaction mixture wasdiluted with MeOH (5 mL) and purified by prep RP-HPLC (0.05% TFA inH₂O:ACN 99:1 to 5:95 over 40 minutes) to give compound 28 as a darkpurple solid (625 mg, 71% yield). MS (ESI) m/z (M+H)⁺ calcd forC₃₉H₄₃FN₄O₉P⁺ 761.3, found 761.3.

Compound 29 is made by the same method as compound 21, and compound 30is made by the same method as compound 37, below.

Compound 32. 4-Hydroxymandelic acid (31) (11.0 g, 59.1 mmol) andN-boc-diaminohexane (14.1 g, 65.0 mmol) were dissolved in DMF (125 mL)followed by sequential addition of HOBt (800 mg, 5.91 mmol), EDAC (17.0g, 88.7 mmol), and finally DIEA (11.5 g, 15.4 mL, 88.7 mmol). Thereaction mixture was stirred under N₂ for 16 hours, followed byreduction of the DMF to about 25 mL under reduced pressure. Theresulting mixture was then quenched with 1M HCl (100 mL) and extractedwith EtOAc (3×100 mL). The organics were collected, combined, and driedover MgSO₄. The solvent was removed under reduced pressure and theresulting residue was purified by flash chromatography in three separateand equal portions (Biotage Snap 50; hex:EA 1:0 to 5:95) to givecompound 32 as a colorless viscous oil (18.7 g, 86% yield). ¹H NMR (400MHz, CDCl₃) δ 8.28 (s, 1H), 7.07 (d, 2H, J=8.4 Hz), 6.81 (br s, 1H),6.64 (d, 2H, J=8.4 Hz), 4.87 (s, 1H), 4.78 (br s, 1H), 4.57 (br s, 1H),3.23-3.11 (m, 2H), 2.99-2.97 (m, 2H), 1.42-1.35 (m, 13H), 1.18 (br s,4H); ¹³C NMR (101 MHz, CDCl₃) δ 173.4, 162.7, 157.0, 128.2, 115.8, 79.4,73.8, 40.34, 39.2, 36.5, 31.5, 29.7, 29.2, 28.4, 26.2, 26.1. MS (ESI)m/z (M+2H-boc)⁺ calcd for C₁₄H₂₃N₂O₃ ⁺ 267.2, found 266.6.

Compound 33. Compound 32 (18.5 g, 50.5 mmol) was dissolved in EtOAc (25mL) and cooled to 0° C. in an ice bath under N₂. Triethylamine (25.6 g,35.2 mL, 253 mmol) was then added and allowed to stir for 10 minutes.Diethyl chlorophosphate (9.15 g, 7.69 mL, 53.0 mmol) was then addeddropwise over a period of 5 minutes. The reaction mixture was removedfrom the ice bath and stirred under N₂ for 4 hours. The reaction mixturewas then quenched with 1M HCl (200 mL) and extracted with EtOAc (3×200mL). The organics were collected, combined, and dried over MgSO₄. Thesolvent was removed under reduced pressure and the resulting residue waspurified by flash chromatography (Biotage Snap 340; hex:EA 1:0 to 5:95)to give compound 33 as a colorless viscous oil (22.6 g, 89% yield). ¹HNMR (400 MHz, CDCl₃) δ 7.36 (d, 2H, J=8.4 Hz), 7.14 (d, 2H, J=8.4 Hz),6.66 (br s, 1H), 4.85 (s, 1H), 4.64 (br s, 2H), 4.20-4.13 (m, 4H),3.24-3.20 (m, 2H), 3.08-3.03 (m, 2H), 1.40-1.25 (m, 23H); ¹³C NMR (101MHz, CDCl₃) δ 172.1, 156.1, 150.5, 150.4, 136.9, 128.1, 120.04, 119.99,79.1, 73.2, 64.8, 64.71, 64.65, 40.1, 39.1, 29.8, 29.2, 28.4, 26.04,25.98, 16.07, 16.00; ³¹P NMR (162 MHz, CDCl₃) δ −5.9. MS (ESI) m/z(M+H)⁺ calcd for C₂₃H₄₀N₂O₈P⁺ 503.5, found 503.2.

Compound 34. Compound 33 (21.5 g, 42.3 mmol) was dissolved in dry CH₂Cl₂(100 mL) followed by cooling to 0° C. in an ice bath under N₂.Deoxo-Fluor® (10.4 g, 8.68 mL, 47.1 mmol) was then added dropwise over aperiod of 15 minutes. The reaction mixture was removed from the ice bathand stirred under N₂ for 1 hour. The reaction mixture was then quenchedwith a saturated NaHCO₃ solution followed by extraction with CH₂Cl₂(3×100 mL). The organics were collected, combined, and dried over MgSO₄.The solvent was removed under reduced pressure and the resulting residuewas purified by flash chromatography (Biotage Snap 340; CH₂Cl₂:MeOH 1:0to 92:8) to give compound 34 as a light yellow viscous oil (14.9 g, 69%yield). ¹H NMR (400 MHz, CDCl₃) δ 7.36 (d, 2H, J=8.4 Hz), 7.17 (d, 2H,J=8.4 Hz), 6.81 (br s, 1H), 5.67 (d, 2H, J=48 Hz), 4.73 (br s, 1H),4.20-4.10 (m, 4H), 3.26-3.20 (m, 2H), 3.05-2.98 (m, 2H), 1.50-1.20 (m,23H); ¹³C NMR (101 MHz, CDCl₃) δ 168.2, 168.0, 155.9, 151.28, 151.25,151.21, 151.18, 131.7, 131.5, 128.03, 127.97, 119.95, 119.90, 91.9,90.0, 78.7, 64.55, 64.48, 40.1, 38.7, 29.7, 29.1, 28.2, 26.07, 25.98,15.90, 15.83; ¹⁹F NMR (376 MHz, CDCl₃) δ −175.5; ³¹P NMR (162 MHz,CDCl₃) δ −6.0. MS (ESI) m/z (M+2H-boc)⁺ calcd for C₁₈H₃₁FN₂O₅P⁺ 405.2,found 405.0.

Compound 35. Compound 34 (3.20 g, 6.34 mmol) was dissolved in dry CHCl₃(20 mL) followed by cooling to 0° C. in an ice bath under N₂. TMSBr(4.85 g, 4.19 mL, 31.7 mmol) was then added dropwise over a period of 10minutes. The reaction mixture was then removed from the ice bath andstirred under N₂ for 16 hours. The reaction mixture was quenched withMeOH (20 mL) and solvents were removed under reduced pressure. Theresidue was taken up in MeOH (10 mL) and the resulting mixture was addeddrop wise to a stirring flask of ice water (100 mL), resulting in athick white precipitate. The solid was collected by vacuum filtrationand washed with cold water. The resulting white solid was dried underhigh vacuum to give compound 35 as a white solid (1.50 g, 68% yield). ¹HNMR (400 MHz, DMSO-d₆) δ 8.32 (t, 1H, J=5.4 Hz), 8.17 (br s, 3H), 7.36(d, 2H, J=8.4 Hz), 7.16 (d, 2H, J=8.4 Hz), 5.72 (d, 2H, J=48 Hz),3.13-3.07 (m, 2H), 2.29 (br s, 2H), 1.35-1.20 (m, 4H), 1.05-1.00 (m,2H), 0.90-0.84 (m, 2H); ¹³C NMR (101 MHz, DMSO-d₆) δ 168.3, 168.1,154.7, 129.6, 129.4, 127.23, 127.18, 91.4, 89.6, 38.3, 37.1, 28.6, 26.9,25.5, 24.9; ¹⁹F NMR (376 MHz, DMSO-d6) δ −174.2; ³¹P NMR (162 MHz,DMSO-d₆) δ −4.8. MS (ESI) m/z (M+H)⁺ calcd for C₁₄H₂₂FN₂O₅P⁺ 349.3,found 348.9.

Examples of Conjugation to Compound 35: Compound 36.5(6)-Carboxytetramethylrhodamine (500 mg, 1.16 mmol) was dissolved indry DMSO (5 mL) followed by addition of DMAP (213 mg, 1.74 mmol) andN,N′-disuccinimidyl carbonate (327 mg, 1.28 mmol). The reaction vesselwas sealed and the reaction mixture stirred at room temperature for 30minutes. Compound 35 (446 mg, 1.28 mmol) was then added, followed byaddition of DIEA (750 mg, 1.01 mL, 5.80 mmol). The resulting mixture wasstirred at room temperature for 2 hours. The reaction mixture wasdiluted with MeOH (5 mL) and purified by prep RP-HPLC (0.05% TFA inH₂O:ACN 99:1 to 5:95 over 40 minutes) to give compound 36 as a darkpurple solid (625 mg, 71% yield). MS (ESI) m/z (M+H)⁺ calcd forC₃₉H₄₃FN₄O₉P⁺ 761.3, found 761.3.

Compound 37. Amino-pegs-acid (511 mg, 1.16 mmol) was added to a solutionof dry DMSO (10 mL) and DIEA (449 mg, 605 μL, 3.47 mmol) followed bysonication until a clear solution was observed. Dabsyl chloride (750 mg,2.32 mmol) was then added in ten equal portions over 15 minutes. Thereaction vessel was sealed and the reaction mixture was stirred at roomtemperature for 1 hour. DMAP (212 mg, 1.74 mmol) was then added,followed by the addition of N,N′-disuccinimidyl carbonate (327 mg, 1.28mmol) in ten equal portions over 15 minutes. The reaction vessel wassealed and the reaction mixture was stirred at room temperature for 15minutes. Compound 35 (446 mg, 1.28 mmol) was then added, followed byaddition of DIEA (750 mg, 1.01 mL, 5.80 mmol). The resulting mixture wasstirred at room temperature for 2 hours. The reaction mixture wasdiluted with MeOH (5 mL) and purified by prep RP-HPLC (0.05% TFA inH₂O:ACN 99:1 to 5:95 over 40 minutes) to give compound 37 as a darkorange viscous oil (735 mg, 60% yield based on amino-pegs-acid). MS(ESI) m/z (M+2H)²⁺ calcd for C₄₇H₇₄FN₆O₁₆PS²⁺ 530.2, found 530.3.

Compound 38. Compound 32 (4.49 g, 12.3 mmol) was dissolved in EtOAc (25mL) followed by addition of imidazole (2.08 g, 30.6 mmol) and TBS-Cl(4.61 g, 30.6 mmol). The reaction vessel was sealed and stirred at roomtemperature for 4 hours. The solvent was then removed under reducedpressure and the resulting residue taken up in a mixture of MeCN:H₂O(10:1, 20 mL). DBU (1.87 g, 12.3 mmol) was then added and the reactionmixture stirred at room temperature for 16 hours. The reaction mixturewas then extracted with EtOAc (50 mL) and 1M HCl (3×50 mL). The organiclayer was collected, dried over MgSO₄, and the solvent removed underreduced pressure. The resulting residue was purified by flashchromatography (Biotage Snap 50; hex:EA 1:0 to 1:4) to give compound 38as a colorless oil (5.10 g, 86% yield). ¹H NMR (400 MHz, CDCl₃) δ 7.89(br s, 1H), 7.06 (d, 2H, J=8.4 Hz), 7.01 (t, 1H, J=5.6 Hz), 6.54 (d, 2H,J=8.4 Hz), 4.95 (s, 1H), 4.62 (br s, 1H), 3.30-3.20 (m, 2H), 3.09-3.01(m, 2H), 1.55-1.46 (m, 13H), 1.28 (s, 4H), 0.88 (s, 9H), 0.05 (s, 3H),−0.12 (s, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 173.3, 156.7, 130.5, 127.6,115.6, 79.2, 75.6, 40.4, 38.8, 29.9, 29.5, 28.4, 26.3, 25.7, 18.1, −4.7,−5.3. MS (ESI) m/z (M+H)⁺ calcd for C₂₅H₄₅N₂O₅Si⁺ 481.3, found 481.3.

Compound 39. Compound 38 (1.75 g, 3.64 mmol), TBABr (2.35 g, 7.28 mmol),and acetobromo-α-D-galactoside (2.99 g, 7.28 mmol) were combined in around bottom flask and dissolved in CH₂Cl₂ (25 mL). An aqueous solutionof NaOH (5% wt, 12 mL) was then added and the reaction mixture stirredvigorously at room temperature for 4 hours. The reaction mixture wasthen diluted with brine (50 mL) and extracted with CH₂Cl₂ (3×50 mL). Theorganic layer was collected, dried over MgSO₄, and the solvent removedunder reduced pressure. The resulting residue was purified by flashchromatography (Biotage Snap 50; hex:EA 1:0 to 1:4) to give compound 39as a colorless oil (2.45 g, 83% yield). ¹H NMR (400 MHz, CDCl₃) δ 7.34(dd, 2H, J=8.8 Hz and 2.0 Hz), 6.93 (d, 2H, J=8.8 Hz), 6.82-6.78 (m,1H), 5.49-5.41 (m, 2H), 5.06 (dd, 1H, J=14 Hz and 3.6 Hz), 5.03-5.00 (m,2H), 4.55 (br s, 1H), 4.25-4.00 (m, 3H), 3.33-3.25 (m, 1H), 3.17-3.02(m, 3H), 2.16 (s, 3H), 2.04 (s, 6H), 1.99 (s, 3H), 1.50-1.40 (m, 13H),1.29 (br s, 4H), 0.91 (s, 9H), 0.06 (s, 3H), −0.06 (s, 3H); ¹³C NMR (101MHz, CDCl₃) δ 171.9, 170.3, 170.2, 170.0, 169.3, 156.66, 156.64, 155.9,134.86, 136.84, 127.34, 127.28, 116.7, 99.60, 99.57, 79.0, 75.1, 75.0,70.92, 70.89, 70.8, 68.6, 66.8, 61.3, 40.3, 38.7, 29.9, 29.5, 28.4,26.4, 26.3, 25.7, 20.7, 20.6, 20.5, 18.1, −4.8, −5.4. MS (ESI) m/z(M+H)⁺ calcd for C₃₉H₆₃N₂O₁₄Si⁺ 811.4, found 811.7.

Compound 40. Compound 39 (2.15 g, 2.65 mmol) was dissolved in THF (20mL) followed by purging with N₂ and cooling to 0° C. in an ice bath.TBAF (1M in THF, 2.65 mL, 2.65 mmol) was then added dropwise over aperiod of 5 minutes. The reaction mixture was stirred at 0° C. under N₂for 15 minutes, followed by quenching with a solution of saturatedNaHCO₃ (25 mL). The resulting suspension was extracted with EtOAc (3×50mL). The organics were combined, dried over MgSO₄, and the solventremoved under reduced pressure. The resulting residue was purified byflash chromatography (Biotage Snap 50; hex:EA 1:0 to 1:9) to givecompound 40 as a colorless oil that became a white foam upon exposure tohigh-vac (1.55 g, 84% yield). ¹H NMR (400 MHz, CDCl₃) D D7.35 (d, 2H,J=8.4 Hz), 6.99 (d, 2H, J=8.4 Hz), 6.36 (d, 1H, J=5.6 Hz), 5.50-5.43 (m,2H), 5.10 (dd, 1H, J=10 Hz and 3.2 Hz), 5.05-4.98 (m, 2H), 4.57 (br s,1H), 4.25-4.02 (m, 3H), 4.04 (br s, 1H), 3.30-3.20 (m, 2H), 3.21-3.03(m, 2H), 2.18 (s, 3H), 2.05-2.00 (m, 9H), 1.53-1.33 (m, 13H), 1.32-1.20(m, 4H); ¹³C NMR (101 MHz, CDCl₃) δ 172.1, 170.4, 170.2, 170.1, 169.4,157.0, 156.1, 134.65, 134.63, 128.2, 117.2, 117.1, 99.62, 99.60, 79.2,73.53, 73.49, 71.0, 70.8, 68.6, 66.8, 61.3, 40.0, 39.2, 29.8, 29.2,28.4, 25.9, 20.70, 20.65, 20.64, 20.56. MS (ESI) m/z (M+H)⁺ calcd forC₃₃H₄₉N₂O₁₄ ⁺ 697.3, found 697.5.

Compound 41. Compound 40 (1.22 g, 1.75 mmol mmol) was dissolved in dryCH₂Cl₂ (20 mL) followed by cooling to 0° C. in an ice bath under N₂.Deoxo-Fluor® (426 mg, 355 μL, 1.93 mmol) was then added dropwise over aperiod of 5 minutes. The reaction mixture was removed from the ice bathand stirred under N₂ for 15 minutes. The reaction mixture was thenquenched with a saturated NaHCO₃ solution (20 mL) followed by extractionwith CH₂Cl₂ (3×50 mL). The organics were collected, combined, and driedover MgSO₄. The solvent was removed under reduced pressure and theresulting residue was purified by flash chromatography (Biotage Snap 50;hex:EA 1:0 to 1:9) to give compound 41 as a colorless oil that became awhite foam upon exposure to high-vacuum (840 mg, 69% yield). ¹H NMR (400MHz, CDCl₃) δ 7.35 (d, 2H, J=8.4 Hz), 6.99 (d, 2H, J=8.4 Hz), 6.63 (brs, 1H), 5.69 (d, 1H, J=5.6 Hz), 5.50-5.41 (m, 2H), 5.08 (dd, 1H, J=10 Hzand 3.6 Hz), 5.03 (d, 1H, J=8.0 Hz), 4.58 (br s, 1H), 4.23-4.01 (m, 3H),4.04 (br s, 1H), 3.30-3.23 (m, 2H), 3.12-3.01 (m, 2H), 2.16 (s, 3H),2.02-1.96 (m, 9H), 1.58-1.25 (m, 17H); ¹³C NMR (101 MHz, CDCl₃) δ 170.3,170.1, 170.0, 169.3, 168.4, 168.2, 162.4, 157.62, 157.60, 157.57, 156.0,129.94, 129.91, 129.75, 129.72, 128.31, 128.24, 128.18, 116.90, 116.89,99.3, 92.27, 92.23, 90.40, 90.37, 79.0, 70.99, 70.98, 70.7, 68.5, 66.8,61.3, 40.2, 38.9, 36.4, 31.2, 29.9, 29.3, 28.3, 26.2, 26.1, 20.62,20.57, 20.48; ¹⁹F NMR (376 MHz, CDCl₃) δ −175.2, −175.6. MS (ESI) m/z(M+Na)⁺ calcd for C₃₃H₄₇FN₂NaO₁₃ ⁺ 721.3, found 721.2.

Compound 42. Compound 41 (300 mg, 0.429 mmol) was dissolved in a mixtureof TEA:MeOH:H₂O (1:8:1, 2 mL) and stirred at room temperature for 4hours. The solvents were removed under reduced pressure followed byaddition of a solution of TFA:CH₂Cl₂ (1:1, 5 mL). The reaction vesselwas sealed and stirred at room temperature for 1 hours. The solventswere removed under reduced pressure and the resulting residue purifiedby prep RP-HPLC (0.05% TFA in H₂O:ACN 99:1 to 5:95 over 60 minutes) togive compound 42 as a white solid (75 mg, 41% yield). ¹H NMR (400 MHz,DMSO-d₆) δ 8.43 (t, 1H, J=5.6 Hz), 7.67 (br s, 3H), 7.35 (d, 2H, J=8.0Hz), 7.05 (d, 2H, J=8.0 Hz), 5.77 (d, 1H, J=48 Hz), 5.18 (s, 1H),4.95-4.80 (m, 2H), 4.67 (s, 1H), 4.53 (s, 1H), 3.70 (s, 1H), 3.60-3.25(m, 7H), 3.20-3.05 (m, 2H), 1.55-1.38 (m, 4H), 1.37-1.20 (m, 4H); ¹³CNMR (101 MHz, DMSO-d₆) δ 168.8, 167.7, 158.0, 129.2, 129.0, 128.6,128.5, 116.1, 100.6, 91.50, 91.45, 89.69, 89.63, 75.5, 73.3, 70.2, 68.1,60.3, 38.8, 38.1, 28.7, 26.9, 25.7, 25.4; ¹⁹F NMR (376 MHz, CDCl₃) δ−170.4, −170.5. MS (ESI) m/z (M+H)⁺ calcd for C₂₀H₃₂FN₂O₇ ⁺ 431.2, found431.1.

Example of Conjugation to Compound 42: Compound 43. Compound 42 (5 mg,12 μmol) was dissolved in dry DMSO (1 mL) followed by addition oftriethylamine (6 mg, 8 μL, 58 μmol) and finally Cy5-NHS ester (7 mg, 12μmol). The reaction vessel was sealed and the reaction mixture stirredat room temperature for 1 hour. The reaction mixture was diluted withMeOH (1 mL) and directly purified by prep RP-HPLC (0.05% TFA in H₂O:ACN99:1 to 5:95 over 40 minutes) to give compound 43 (TFA salt) as a bluesolid (8 mg, 68% yield). MS (ESI) m/z (M)⁺ calcd for C₅₂H₆₈FN₄O₈ ⁺895.5, found 895.2.

Compound 44. Compound 32 (200 mg, 0.546 mmol) was dissolved in EtOAc (1mL) followed by addition of triethylamine (166 mg, 1.64 mmol) andpropionyl chloride (56 mg, 0.600 mmol) in a round bottom flask. Thereaction mixture was stirred at room temperature for 1 hour. Thereaction mixture was then extracted with 0.5M HCl (5 mL) and EtOAc (3×10mL), the organic layer collected and dried over MgSO₄. The suspensionwas filtered, the filtrate collected and the solvents removed underreduced pressure. The resulting residue was purified by flashchromatography (hex:EtOAc 1:0 to 1:9) to give compound 44 as a whitesolid (210 mg, 91% yield): ¹H NMR (400 MHz, CD₃CN) δ 7.42 (d, 2H, J=8.4Hz), 7.14 (br s, 1H), 7.06 (d, 2H, J=8.4 Hz), 5.34 (br s, 1H), 4.96 (d,1H, J=4.0 Hz), 4.51 (d, 1H, J=4.0 Hz), 3.15 (q, 2H, J=6.8 Hz), 2.97 (q,2H, J=6.8 Hz), 2.58 (q, 2H, J=7.6 Hz), 1.42-1.30 (m, 13H), 1.29-1.12 (m,7H); ¹³C NMR (101 MHz, CD₃CN) δ 174.1, 173.0, 157.0, 151.6, 139.5,128.8, 122.7, 78.98, 74.2, 41.0, 39.6, 30.7, 30.2, 28.7, 28.2, 27.07,27.03, 9.36. MS (ESI) m/z (M+Na)⁺ calcd for C₂₂H₃₄N₂NaO₆ ⁺ 445.2, found445.2.

Compound 45. Compound 44 (200 mg, 0.473 mmol) was dissolved in CHCl₃ (5mL) in a sealed scintillation vial and cooled to 0° C. in an ice bath.Deoxo-Fluor® (110 mg, 0.497 mmol) was then added drop wise and thereaction vessel sealed. The reaction mixture was stirred at 0° C. for 1hour, followed by quenching with 0.5 M HCl (5 mL). The organic layer wasseparated and dried over MgSO₄. The suspension was filtered, thefiltrate collected and the solvents removed under reduced pressure. Theresulting residue was purified by flash chromatography (hex:EtOAc 1:0 to1:9) to give compound 45 as a white solid (155 mg, 77% yield): ¹H NMR(400 MHz, CDCl₃) δ 7.39 (d, 2H, J=8.4 Hz), 7.04 (d, 2H, J=8.4 Hz), 6.81(br s, 1H), 5.68 (d, 1H, J=48 Hz), 4.77 (br s, 1H), 3.21 (q, 2H, J=6.0Hz), 3.00-2.95 (m, 2H), 2.52 (q, 2H, J=7.6 Hz), 1.42-1.30 (m, 13H),1.29-1.11 (m, 7H); ¹³C NMR (101 MHz, CDCl₃) δ 172.5, 168.1, 167.9,155.9, 151.19, 151.17, 132.4, 132.2, 127.53, 127.47, 121.5, 91.9, 90.0,78.6, 40.02, 38.65, 29.7, 29.1, 28.2, 27.4, 26.0, 25.9, 8.7; ¹⁹F NMR(376 MHz, CDCl₃) δ −178.0. MS (ESI) m/z (M+Na)⁺ calcd for C₂₂H₃₃FN₂NaO₅⁺ 447.2, found 447.1.

Compound 46. Compound 45 (100 mg, 0.236 mmol) was dissolved in a 1:1mixture of CH₂Cl₂:TFA (1 mL) in a scintillation vial. The vial wassealed and stirred at room temperature for 30 minutes. The solvents werethen removed under reduced pressure and the resulting residue was foundto be of suitable purity for subsequent synthetic steps. Compound 46 wasobtained as a viscous oil determined to be the TFA salt (100 mg, 97%yield). A small sample was purified by prep RP-HPLC (0.05% TFA inH₂O:ACN 99:1 to 5:95 over 40 minutes) to obtain an analytical sample. ¹HNMR (400 MHz, CDCl₃) δ 7.42 (d, 2H, J=7.6 Hz), 7.09 (d, 2H, J=7.6 Hz),6.98 (br s, 1H), 5.72 (d, 1H, J=48 Hz), 3.23 (br s, 2H), 2.57 (q, 2H,J=7.6 Hz), 1.60-1.40 (m, 4H), 1.39-1.15 (m, 7H); ¹³C NMR (101 MHz,CDCl₃) δ 173.1, 169.0, 168.8, 151.52, 151.50, 132.5, 132.3, 128.0,127.9, 121.9, 91.9, 90.1, 39.6, 38.7, 28.7, 27.6, 26.9, 25.5, 25.2, 8.9;¹⁹F NMR (376 MHz, CDCl₃) δ −177.5. MS (ESI) m/z (M+H)⁺ calcd forC₁₇H₂₆FN₂O₃ ⁺ 325.2, found 325.2.

Example of Conjugation to Compound 46: Compound 47. Compound 46 (TFAsalt, 10 mg, 23 μmol) was dissolved in dry DMSO (1 mL) followed byaddition of triethylamine (7 mg, 10 μL, 68 μmol) and finally Cy5-NHSester (15 mg, 25 μmol). The reaction vessel was sealed and the reactionmixture stirred at room temperature for 1 hour. The reaction mixture wasdiluted with MeOH (1 mL) and directly purified by prep RP-HPLC (0.05%TFA in H₂O:ACN 99:1 to 5:95 over 40 minutes) to give compound 47 (TFAsalt) as a blue solid (12 mg, 58% yield). MS (ESI) m/z (M)⁺ calcd forC₄₉H₆₂FN₄O₄ ⁺ 789.5, found 789.1.

Resorufin is treated with sodium hydroxide in chloroform and water toform aldehyde 48. Aldehyde 48 is then reacted with compound 20 in thepresence of a suitable reducing agent, such as sodium cyanoborohydride,to form compound 41.

Synthesis of QMs containing 1,2,3-triazolyl linker by azide-alkyne“click” chemistry Compound 57 is prepared by the reaction sequence shownin Scheme 11. First, salicylaldehyde (50) is reacted withethynylmagnesium bromide (51) to give compound 52. Then, compound 52 isreacted in sequence with diethylchlorophosphate (53) and Deoxofluor® togive compound 54. Compound 54 is deprotected with TMSBr to give the arylphosphate 55. Finally, compound 55 is reacted with terminalazide-functionalized reporter molecules (N₃—R⁴, compound 56) to give theQM-reporter conjugates containing a 1,2,3-triazolyl linker (57).

Example 2 Target Detection Using QMPs

General Immunohistochemistry (IHC) Protocol(s) for QMPs. All IHCstaining experiments were carried out on a VENTANA BenchMark® XTautomated tissue staining platform and the reagents used in theseprotocols were from Ventana Medical Systems, Inc. (Tucson, AZ, USA;“Ventana”) unless otherwise specified. Polyclonal goat anti-rabbitantibodies, goat anti-mouse antibodies, horseradish peroxidase (HRP) andalkaline phosphatase (AP) were obtained from Roche Diagnostics(Mannheim, Germany).

The following common steps were performed: (1) deparaffinization with EZPrep detergent solution (Ventana Medical Systems, Inc. (VMSI), #950-101)(75° C.; 20 minutes); (2) washing with Reaction Buffer (VMSI, #950-300);(3) antigen retrieval in Cell Conditioning 1 (VMSI #950-124) (100° C.;time dependent on antigen of interest); (4) washing (same as step 2);(5) for protocols with subsequent HRP detection steps endogenousperoxidase was inactivated using iVIEW inhibitor (VMSI, E253-2187) (37°C.; 4 minutes); (6) washing (same as step 2); (7) primary antibody (Ab)incubation (37° C.; time dependent on primary antibody ranging from 8-32minutes); and (8) washing (same as step 2). All subsequent reagentincubation steps were separated by washing as in step (2).

Two experimental staining protocols were used, as illustrated in FIGS. 4and 5 Primary antibody (anti-target antibody) incubation and washingwere followed by secondary antibody incubation with a goat polyclonalanti-species antibody conjugated to AP (37° C.; 8 minutes) (FIG. 4 ).Primary antibody incubation and washing were followed by secondaryantibody incubation with a goat polyclonal anti-species antibody, haptenlabeled with nitropyrazole (NP) (37° C.; 8 minutes). After washing,AP-conjugated mouse anti-NP monoclonal antibody was added (37° C.; 8minutes) (FIG. 5 ).

Detection 1—HRP DAB. After incubating with the AP conjugate the slideswere washed with Special Stains wash (VMSI #860-015). QMP reagents weredissolved in 100 mM CHES (3-cyclo-hexylamino-ethylsulfonic acid), pH10.0, 0.05% Brij-35. QMP turnover was achieved by adding 100 μL of APEnhancer (VMSI #253-2182) followed by 100 μL of hapten labeled QMP andincubating at 37° C. for 16 minutes. Compound 29 is an exemplaryhapten-QMP that could be used according to this method. The depositedhapten was subsequently bound by a mouse-anti-hapten-HRP conjugate (orstreptavidin-HRP) (37° C.; 8 minutes), and visualized via a brownprecipitate produced by HRP upon the addition of hydrogen peroxide andDAB (37° C.; 8 minutes). The DAB was toned by the addition of coppersulfate (37° C.; 4 minutes). The stained tissue sections werecounterstained with modified Mayer's hematoxylin (Hematoxylin II, VMSI#790-2208) (37° C.; 4 minutes) and then incubated with Bluing Reagent(VMSI #760-2037) (37° C.; 4 minutes). The slides were then dehydratedthrough a graded ethanol series, cleared with xylene, and manuallycover-slipped. FIG. 13(A) shows an exemplary microphotograph of a slidestained according to this method.

Detection 2—AP Red. After incubating with the AP conjugate the slideswere washed with Special Stains wash. QMP reagents were dissolved in 100mM CHES, pH 10.0, 0.05% Brij-35. QMP turnover was achieved by adding 100μL of AP Enhancer followed by 100 μL of hapten labeled QMP andincubating at 37° C. for 16 minutes. Compound 29 is an exemplaryhapten-QMP that could be used according to this method. The depositedhapten was subsequently bound by a mouse-anti-hapten-AP conjugate (orstreptavidin-AP conjugate) (37° C.; 8 minutes), and detection wasachieved by adding 100 μL of AP Enhancer, followed by 100 μL of NaphtholAS-TR Phosphate and 200 μL of Fast Red KL (37° C.; 16 minutes). Thestained tissue sections were counterstained with modified Mayer'shematoxylin (37° C.; 4 minutes) and then incubated with Bluing Reagent(37° C.; 4 minutes). The slides were rinsed with a detergent watermixture, air dried and manually cover-slipped. FIGS. 14(B)-14(D) showexemplary microphotographs of slides stained according to this method.

Detection 3—Quantum Dot. After incubating with the AP conjugate theslides were washed with Special Stains wash. QMP reagents were dissolvedin 100 mM CHES, pH 10.0, 0.05% Brij-35. QMP turnover was achieved byadding 100 μL of AP Enhancer followed by 100 μL of hapten labeled QMPand incubating at 37° C. for 16 minutes. Compound 29 is an exemplaryhapten-QMP that could be used according to this method. The depositedhapten was subsequently visualized by incubation with amouse-anti-hapten quantum dot conjugate (or streptavidin quantum dotconjugate) (37° C.; 32 minutes). The slides were washed with ReactionBuffer, dehydrated through a graded ethanol series, cleared with xylene,and manually cover-slipped. FIG. 15C shows an exemplary microphotographof a slide stained according to this method.

Detection 4—Fluorophore. After incubating with the AP conjugate theslides were washed with saline sodium citrate buffer (SSC, VMSI#950-110). QMP reagents were dissolved in 250 mM Tris, pH 10.0, 0.05%Brij-35. QMP turnover was achieved by adding 100 μL of AP Enhancerfollowed by 100 μL of fluorophore labeled QMP (at a concentration<50 μM)and incubating at 37° C. for 16 minutes. Compound 28 is an exemplaryfluorophore-QMP that could be used according to this method. The slideswere washed with Reaction Buffer, dehydrated through a graded ethanolseries, cleared with xylene, and manually cover-slipped. The slides wereviewed by fluorescence microscopy using the appropriate filter sets.FIG. 15A shows an exemplary microphotograph of a slide stained accordingto this method

Detection 5—Chromogenic QMP. After incubating with the AP conjugate theslides were washed with SSC. QMP reagents were dissolved in 250 mM Tris,pH 10.0, 0.05% Brij-35. QMP turnover was achieved by adding 100 μL of APEnhancer followed by 100 μL of chromophore labeled QMP (at aconcentration>50 PM) and incubating at 37° C. for 16 minutes. Compound30 is an exemplary chromogen-QMP that could be used according to thismethod. In some cases the stained tissue sections were counterstainedwith modified Mayer's hematoxylin (37° C.; 4 minutes) and then incubatedwith Bluing Reagent (37° C.; 4 minutes). The slides were rinsed with adetergent water mixture, then dehydrated through a graded ethanolseries, cleared with xylene, and manually cover-slipped. The slides wereviewed by brightfield microscopy. FIG. 16(A) shows an exemplarymicrophotograph of a slide stained according to this method.

Example 3 HRP DAB Amplified by Difluoro QMP-Biotin (FIGS. 17(A)-(B))

(a) ultraView Control (FIG. 17(A)). The tissue was deparaffinized asdescribed in the general procedures, followed by antigen retrieval withProtease 1 (VMSI #760-2018) (37° C., 8 minutes). Mouse-anti-EGFRantibody incubation (37° C., 32 minutes) and washing were followed bysecondary antibody incubation with a goat polyclonal anti-mouse antibodyconjugated to HRP (37° C.; 8 minutes). The antigen was visualized via abrown precipitate produced by HRP upon the addition of hydrogen peroxideand DAB (37° C.; 8 minutes). The DAB was toned by the addition of coppersulfate (37° C.; 4 minutes). The stained tissue sections werecounterstained with modified Mayer's hematoxylin (37° C.; 4 minutes) andthen incubated with Bluing Reagent (37° C.; 4 minutes) to change thehematoxylin hue to blue. They were then dehydrated through a gradedethanol series, cleared with xylene, and manually cover-slipped.

(b) QMP amplified DAB (FIG. 17(B)). The tissue was deparaffinized asdescribed in the general procedures, followed by antigen retrieval withProtease 1 (37° C., 8 minutes). Mouse-anti-EGFR antibody incubation (37°C., 32 minutes) and washing were followed by secondary antibodyincubation with a goat polyclonal anti-mouse antibody conjugated to AP(37° C.; 12 minutes). After incubating with the AP conjugate the slideswere washed with Special Stains wash. Difluoro QMP-biotin was dissolvedin 100 mM CHES, pH 10.0, 0.05% Brij-35 to a final concentration of 100nM. QMP turnover was achieved by adding 100 μL of AP Enhancer followedby 100 μL of biotin labeled QMP and incubating at 37° C. for 16 minutes.The deposited hapten was subsequently bound by streptavidin-HRPconjugate (37° C.; 8 minutes), and visualized via a brown precipitateproduced by HRP upon the addition of hydrogen peroxide and DAB (37° C.;8 minutes). The DAB was toned by the addition of copper sulfate (37° C.;4 minutes). The stained tissue sections were counterstained withmodified Mayer's hematoxylin (37° C.; 4 minutes) and then incubated withBluing Reagent (37° C.; 4 minutes). They were then dehydrated through agraded ethanol series, cleared with xylene, and manually cover-slipped.

In evaluating QM precursors for IHC staining, it was found that theidentity of the leaving group influenced the activity of the QMP. Twophosphate-protected ortho-QM precursors were prepared, based on4-nitrosalicylaldehyde starting material, containing benzyl monofluoro(Scheme 1, compound 7) and benzyl difluoro (compound 58)functionalities:

Both compounds utilize fluoride as the leaving group, but the reactivityof the QM derived from difluoro compound 58 should be considerably lowerthan monofluoro 7 due to the electronic stabilization offered by thegeminal fluorine atom. In fact, previous reports have suggested QMsderived from some monofluoro precursors may be excessively reactive asprobes of enzyme activity, resulting in active site labeling andsubsequent enzyme inhibition. Therefore, the difluoro QM precursorcompound 58 was evaluated first for IHC staining performance on FFPEtissue using the nuclear marker BCL6 on FFPE tonsil tissue as a modelsystem. The biotin-labeled difluoro QM precursor successfully bound tothe sample as evidenced by subsequent biotin visualization usingdiaminobenzidine (DAB) detection. FIG. 18 provides microphotographsillustrating the detection level at varying concentrations of thedifluoro QM precursor in a Tris buffer at pH=8.5 (panel A—DAB control,panel B—1 μM, panel C—10 μM and panel D—20 μM). In addition, significantamplification of signal was observed compared to the DAB control samplewhen on-slide concentrations of difluoro QM precursor compound 58greater than 20 μM were utilized. However, increased diffusion of signalwas also observed in all cases, resulting in considerable, non-desirableoff-target staining.

It was suggested that the diffusion of signal by the difluoro QMprecursor may have arisen from a combination of two kinetic factors: (1)the rate of leaving group ejection and subsequent QM formation after thecleavage of the phosphate group; and (2) the rate of QM quenching,either by a nucleophile on the tissue or in the reaction media. In thecase of the difluoro QM precursor compound 58, the germinal fluorineatom provided stabilization that may have decelerated both factors,resulting in unacceptable diffusion from the target site. It washypothesized that by increasing the pH of the reaction media, the ratesof both QM formation and quenching may be accelerated, leading to betterstaining results. For (1), a more alkaline pH would increase thepopulation of deprotonated phenol after phosphate cleavage, thereforeencouraging fluoride ejection and QM formation. For (2), increased pHwould increase the population of available quenchers from both the waterand the buffer (Tris), effectively decreasing the distance the QMs wouldbe able to diffuse from the target before reaction with a nucleophile.

To test the effect of pH on signal diffusion, difluoro QM precursorcompound 58 was deposited as before at various pH levels within theworking range for AP (7-12 with two shoulders of maximal activity atpH=8.5 and 11). The nuclear marker BCL6 on FFPE tonsil tissue was againchosen as the model. Positive staining was observed across the entire pHrange, although a balance between overall signal and diffusion was seen.FIG. 19 , panels A-H provide microphotographs of the detection levels of20 D M QM precursor in Tris buffer with varying pH: panel A—DAB control;panel B pH=7.0; panel C pH=8.0; panel D pH=8.5; panel E pH=9.0; panel FpH=10.0; panel G pH=11.0; panel H pH=12.0. The diffusion appears todecrease with increasing pH. At a pH of 7, almost no on-target stainingwas observed with a nearly homogeneous signal seen across the entiretissue section. As pH rose, the off-target staining gradually decreased,although overall signal diffusion and off-target staining remainedsignificant even at a pH of 12. The level of amplification increasedonly as the pH rose to 8.5 and then decreased gradually over the rest ofthe range. This effect may be due to a combination of enzyme activityand the population of QM quenchers in the reaction media. At pH levelswell below the optimal AP reaction conditions (7.0), the activity of APwas depressed, resulting in low signal. As the pH increased (8.0-8.5)but remained near the pKa of Tris (8.1), the activity of AP increased ata faster rate than the population of quenchers in the reaction media,leading to a significant increase in signal. Raising the pH further(9-10) increased the quencher population while decreasing AP activity,resulting in a decrease of signal. Although AP had maximal activity atpH 11 in Tris, a slight decrease in staining intensity was observed,most likely due to the excessively high population of quenchers thatcould not be overcome by the increased AP activity (raising the pH from8.5 to 11 increases [Tris base] about 50%, with AP activity increasingby only about 20%). It was determined that even at the upper end of thepH range, the diffusion from the difluoro QM precursor compound 58 wasstill too great to be of clinical use for IHC. This is in contract toBobrow's disclosure, which suggested that a difluoro based QMP wassuitable for use as an IHC reagent.

It was suggested that by utilizing the less stable monofluoro QMprecursor 7, diffusion may be reduced for reasons described above. IHCstaining was first carried out under typical AP IHC condition (pH 8.5 inTris buffer) across a wide concentration range using the nuclear markerBCL6 on FFPE tonsil tissue to determine the optimal concentration ofmonofluoro QM precursor 7 (data not shown). Surprisingly, a much lowerconcentration of monofluoro QM precursor 7 (250 nM) was required to givethe desired level of amplification when compared to difluoro QMprecursor 58 (20 PM). In addition, overall diffusion was greatly reducedcompared to difluoro QM precursor 58, although some diffusion andoff-target staining were still evident at pH=8.5. In an effort to reducediffusion and off-target staining, a pH range (7-12) was tested in Trisbuffer (FIG. 20 : panel A—DAB control; panel B—pH=7.0; panel C—pH=8.0;panel D—pH=8.5; panel E pH=9.0; panel F pH=10.0; panel G—pH=11.0; panelH—pH=12.0). At low pH (7.0-8.0), significant diffusion was seen,creating a stain similar to those observed with the difluoro QMprecursor 58. However, diffusion and off-target staining was graduallyreduced as pH rose, without an unacceptable decrease in overall signal.At a pH of 10, diffusion and off-target staining were nearly eliminated,producing a stain with a high level of signal along with comparablespecificity to the DAB control. Increasing the pH further to 11-12resulted in slightly cleaner stains as evidenced by the more vibrantblue hematoxylin counterstain. However, a slight reduction in overallsignal was observed at these higher pH levels.

Example 4 HRP DAB Amplified by QMP-Biotin with Quaternary Amine LeavingGroups (FIGS. 21(A)-(D))

(a) ultraView Control (FIG. 21(A)). The tissue was deparaffinized andretrieved as described in the general procedures. Rabbit-anti-Ki-67antibody incubation (37° C., 16 minutes) and washing were followed bysecondary antibody incubation with a goat polyclonal anti-rabbitantibody conjugated to HRP (37° C.; 8 minutes). The antigen wasvisualized via a brown precipitate produced by HRP upon the addition ofhydrogen peroxide and DAB (37° C.; 8 minutes). The DAB was toned by theaddition of copper sulfate (37° C.; 4 minutes). The stained tissuesections were counterstained with modified Mayer's hematoxylin (37° C.;4 minutes) and then incubated with Bluing Reagent (37° C.; 4 minutes) tochange the hematoxylin hue to blue. They were then dehydrated through agraded ethanol series, cleared with xylene, and manually cover-slipped.

(b) Quinone methide analog precursor amplified DAB (FIGS. 21(B)-(D)).The tissue was deparaffinized and retrieved as described in the generalprocedures. Rabbit-anti-Ki-67 antibody incubation (37° C., 16 minutes)and washing were followed by secondary antibody incubation with a goatpolyclonal anti-rabbit antibody conjugated to AP (37° C.; 12 minutes).After incubating with the AP conjugate the slides were washed withSpecial Stains wash. Pyridine QMP-biotin (100 μM) (FIG. 21(B)) or DABCOQMP-biotin (100 μM) (FIG. 21(C)) or triethylamine QMP-biotin (100 μM)(FIG. 21(D)) was dissolved in 100 mM CHES, pH 10.0, 0.05% Brij-35 to afinal concentration of 100 nM. QMP turnover was achieved by adding 100μL of AP Enhancer followed by 100 μL of biotin labeled QMP andincubating at 37° C. for 16 minutes. The deposited hapten wassubsequently bound by streptavidin-HRP conjugate (37° C.; 8 minutes),and visualized via a brown precipitate produced by HRP upon the additionof hydrogen peroxide and DAB (37° C.; 8 minutes). The DAB was toned bythe addition of copper sulfate (37° C.; 4 minutes). The stained tissuesections were counterstained with modified Mayer's hematoxylin (37° C.;4 minutes) and then incubated with Bluing Reagent (37° C.; 4 minutes).They were then dehydrated through a graded ethanol series, cleared withxylene, and manually cover-slipped.

These results demonstrate the sub-optimal performance of quaternaryamines as LG for this application, when compared to the control. Thepyridine, DABCO and triethylamine QMP-biotin derivatives all show muchlower staining intensity and greatly diffuse signals (FIGS. 21(B)-(D))compared to the control slide (FIG. 21(A)).

Example 5 HRP DAB Amplified by QMP-Biotin with Various Leaving Groups(FIGS. 22(A)-(D)

(a) ultraView Control (FIG. 22(A)). See Example 4(a).

(b) QMP amplified DAB (FIGS. 22(B)-22(D)). The tissue was deparaffinizedand retrieved as described in the general procedures. Rabbit-anti-Ki-67antibody incubation (37° C., 16 minutes) and washing were followed bysecondary antibody incubation with a goat polyclonal anti-rabbitantibody conjugated to AP (37° C.; 12 minutes). After incubating withthe AP conjugate the slides were washed with Special Stains wash.Monofluoro QMP-biotin (400 nM) (FIG. 22(B)) or acetate QMP-biotin (100μM) (FIG. 22(C)) or methoxy QMP-biotin (100 μM) (FIG. 22(D)) wasdissolved in 100 mM CHES, pH 10.0, 0.05% Brij-35 to a finalconcentration of 100 nM. QMP turnover was achieved by adding 100 μL ofAP Enhancer followed by 100 μL of biotin labeled QMP and incubating at37° C. for 16 minutes. The deposited biotin was subsequently bound by astreptavidin-HRP conjugate (37° C.; 8 minutes), and visualized via abrown precipitate produced by HRP upon the addition of hydrogen peroxideand DAB (37° C.; 8 minutes). The DAB was toned by the addition of coppersulfate (37° C.; 4 minutes). The stained tissue sections werecounterstained with modified Mayer's hematoxylin (37° C.; 4 minutes) andthen incubated with Bluing Reagent (37° C.; 4 minutes). They were thendehydrated through a graded ethanol series, cleared with xylene, andmanually cover-slipped.

Two sub-optimal and one optimal LG's are shown in this example. TheQMP-biotin with an acetate LG (FIG. 22(C)) generates weak, very diffusesignal and the QMP-biotin with a methoxy LG shows well resolved signalbut with low intensity (FIG. 22(D)). The monofluoro LG derivative ofQMP-biotin ((FIG. 22(B)) demonstrates well resolved signal with greateror equal intensity to the control slide (FIG. 22(A)). Of all the LGgroups evaluated the monofluoro gives the best performance.

Example 6 HRP DAB Amplified by QMP-Biotin with Different Linkers (FIGS.23(A)-(C))

The tissue was deparaffinized and retrieved as described in the generalprocedures. Mouse-anti-Bcl-2 antibody incubation (37° C., 16 minutes)and washing were followed by secondary antibody incubation with a goatpolyclonal anti-mouse antibody conjugated to AP (37° C.; 12 minutes).After incubating with the AP conjugate the slides were washed withSpecial Stains wash. Monofluoro QMP-biotin (with aniline amide (FIG.23(A)), benzoic amide (FIG. 23(B)) or tyramide amide linker (FIG. 23(C))(100 nM) was dissolved in 100 mM CHES, pH 10.0, 0.05% Brij-35 to a finalconcentration of 100 nM. QMP turnover was achieved by adding 100 μL ofAP Enhancer followed by 100 μL of biotin labeled QMP and incubating at37° C. for 16 minutes. The deposited biotin was subsequently bound by astreptavidin-HRP conjugate (37° C.; 8 minutes), and visualized via abrown precipitate produced by HRP upon the addition of hydrogen peroxideand DAB (37° C.; 8 minutes). The DAB was toned by the addition of coppersulfate (37° C.; 4 minutes). The stained tissue sections werecounterstained with modified Mayer's hematoxylin (37° C.; 4 minutes) andthen incubated with Bluing Reagent (37° C.; 4 minutes). They were thendehydrated through a graded ethanol series, cleared with xylene, andmanually cover-slipped.

This experiment demonstrates the subtle effect of mildly electronwithdrawing groups (EWG) and electron donating groups (EDG) on thefunctional staining performance of the QMP-biotin. The benzoic amidelinker (Compound 21, FIG. 2(A)) is mildly EWG and results in morediffuse, less resolved signal. The other linkers which are neutral(aniline amide (Compound 7, FIG. 2(A))) or mildly EDG (tyramide amide(Compound 14, FIG. 2(A))) generate well resolved signals. Otherexperiments (not included here) with strong EDG (e.g. methoxy) or EWG(e.g. nitro) show much more deleterious effect on staining performance.This illustrates the influence of electronics on the reactivity of theQMP intermediates and the effect on staining performance.

Example 7 AP Fast Red Amplified by Monofluoro-QMP-NP (FIGS. 14A-(D))

Fast Red control (FIG. 14(A)). The tissue was deparaffinized andretrieved as described in the general procedures. Rabbit-anti-CD-10antibody incubation (37° C., 16 minutes) and washing were followed byincubation with a goat-anti-rabbit polyclonal antibody, hapten labeledwith nitropyrazole (NP) (37° C.; 8 minutes). After washing AP-conjugatedmouse anti-NP monoclonal antibody was added (37° C.; 12 minutes).Detection was achieved by adding 100 μL of AP Enhancer, followed by 100μL of naphthol AS-TR phosphate and 200 μL of Fast Red KL (37° C.; 16minutes). The stained tissue sections were counterstained with modifiedMayer's hematoxylin (37° C.; 4 minutes) and then incubated with BluingReagent (37° C.; 4 minutes). The slides were rinsed with a detergentwater mixture, air dried and manually cover-slipped.

QMP amplified Fast Red (FIGS. 14(B)-14(D)). The tissue wasdeparaffinized and retrieved as described in the general procedures.Rabbit-anti-CD-10, mouse-anti-Bcl-2 or mouse-anti-Her3 antibodyincubation (37° C., 32 minutes) and washing were followed by incubationwith a goat polyclonal anti-rabbit or anti-mouse antibody, haptenlabeled with nitropyrazole (NP) (37° C.; 8 minutes). After washingAP-conjugated mouse anti-NP monoclonal antibody was used as a tertiaryantibody (37° C.; 12 minutes). After incubating with the AP conjugatethe slides were washed with Special Stains wash. Monofluoro QMP-NP (100nM) was dissolved in 100 mM CHES, pH 10.0, 0.05% Brij-35 to a finalconcentration of 100 nM. QMP turnover was achieved by adding 100 μL ofAP Enhancer followed by 100 μL of NP labeled QMP and incubating at 37°C. for 16 minutes. The deposited hapten was subsequently bound by amouse-anti-hapten-AP conjugate (37° C.; 12 minutes), and detection wasachieved by adding 100 μL of AP Enhancer, followed by 100 μL of naphtholAS-TR phosphate and 200 μL of Fast Red KL (37° C.; 16 minutes). Thestained tissue sections were counterstained with modified Mayer'shematoxylin (37° C.; 4 minutes) and then incubated with Bluing Reagent(37° C.; 4 minutes). The slides were rinsed with a detergent watermixture, air dried and manually cover-slipped.

This example demonstrates than once a reporter (in this case the NPhapten) is deposited by QMP methodology then an alternate detectionsystem to DAB can also be used. In this case an anti-NP AP conjugate isused to visualize the target with AP Fast Red chemistry. FIG. 14(B)shows a significant amplification in signal over the control slide (FIG.14(A)). FIGS. 14(C) and 14(D) show strong intensity staining fornormally weakly visualized biomarkers Bcl2 and Her3.

Example 8 Monofluoro-QMP-Fluorophore and Quantum Dot Amplified byMonofluoro-QMP-NP (FIGS. 15(A)-(D))

(a) QMP fluorophore (FIGS. 15(A), 15(B)). The tissue was deparaffinizedand retrieved as described in the general procedures. Mouse-anti-Bcl-2antibody incubation (37° C., 16 minutes) and washing were followed bysecondary antibody incubation with a goat polyclonal anti-mouseantibody, hapten labeled with nitropyrazole (NP) (37° C.; 8 minutes).After washing AP-conjugated mouse anti-NP monoclonal antibody was added(37° C.; 12 minutes). After incubating with the AP conjugate the slideswere washed with Special Stains wash. Monofluoro QMP-TAMRA (FIG. 15(A))or monofluoro QMP-Alexa Fluor© 700 (FIG. 15(B)) was dissolved in 100 mMCHES, pH 10.0, 0.05% Brij-35. QMP turnover was achieved by adding 100 μLof AP Enhancer followed by 100 μL of fluorophore labeled QMP andincubating at 37° C. for 16 minutes. The slides were washed withReaction Buffer, dehydrated through a graded ethanol series, clearedwith xylene, and manually cover-slipped. The slides were viewed byfluorescence microscopy using the appropriate filter sets.

(b) QMP amplified Quantum Dot (FIG. 15(C)). The tissue wasdeparaffinized and retrieved as described in the general procedures.Mouse-anti-Bcl-2 antibody incubation (37° C., 16 minutes) and washingwere followed by secondary antibody incubation with a goat polyclonalanti-mouse antibody, hapten labeled with nitropyrazole (NP) (37° C.; 8minutes). After washing AP-conjugated mouse anti-NP monoclonal antibodywas added (37° C.; 12 minutes). After incubating with the AP conjugatethe slides were washed with Special Stains wash. Monofluoro QMP-NP wasdissolved in 100 mM CHES, pH 10.0, 0.05% Brij-35. QMP turnover wasachieved by adding 100 μL of AP Enhancer followed by 100 μL of NPlabeled QMP and incubating at 37° C. for 16 minutes. The depositedhapten was subsequently visualized by incubation with a mouse-anti-NPQuantum Dot 525 conjugate (37° C.; 32 minutes). The slides were washedwith Reaction Buffer, dehydrated through a graded ethanol series,cleared with xylene, and manually cover-slipped. The slides were viewedby fluorescence microscopy using the appropriate filter sets.

When fluorophores are used as the reporter in a QMP molecule (FIGS.15(A) and 15(B)) then the results can be visualized with any furtherdetection chemistries generating an amplified fluorescent signal. QMP-NPcan be detected fluorescently using a streptavidin-quantum conjugate.This offers an alternative way of detecting the deposited haptencompared to the HRP/DAB method outlined in Examples 3-6. Thisillustrates that the QMP methodology can be used to generate bothbrightfield and darkfield (fluorescent) amplified signals and is easilyadapted to work with existing detection chemistries.

Example 9 HRP DAB Amplified by QMP-NP (FIGS. 13(A)-(B), 24(A)-(B),25(A)-(B))

(a) ultraView Control (FIG. 13(B), FIGS. 24(A)-24(B); 24(B) shows theregion of 24(A) demarcated by a black box at higher magnification). SeeExample 4(a).

QMP amplified DAB (FIG. 13(A), FIGS. 25(A)-25(B); 25(B) shows the regionof 25(A) demarcated by a black box at higher magnification). The tissuewas deparaffinized and retrieved as described in the general procedures.Rabbit-anti-Ki-67 antibody incubation (37° C., 16 minutes) and washingwere followed by secondary antibody incubation with a goat polyclonalanti-rabbit antibody conjugated to AP (37° C.; 12 minutes). Afterincubating with the AP conjugate the slides were washed with SpecialStains wash. Monofluoro QMP-NP (100 nM) was dissolved in 100 mM CHES, pH10.0, 0.05% Brij-35 to a final concentration of 100 nM. QMP turnover wasachieved by adding 100 μL of AP Enhancer followed by 100 μL of NPlabeled QMP and incubating at 37° C. for 16 minutes. The depositedhapten was subsequently bound by a mouse-anti-NP HRP conjugate (37° C.;8 minutes), and visualized via a brown precipitate produced by HRP uponthe addition of hydrogen peroxide and DAB (37° C.; 8 minutes). The DABwas toned by the addition of copper sulfate (37° C.; 4 minutes). Thestained tissue sections were counterstained with modified Mayer'shematoxylin (37° C.; 4 minutes) and then incubated with Bluing Reagent(37° C.; 4 minutes). They were then dehydrated through a graded ethanolseries, cleared with xylene, and manually cover-slipped.

This example demonstrates the amplification of signal using QMP-NPfollowed by HRP/DAB detection. While the signal intensity is greater, noadditional background is generated and the signal localization isequivalent to the control slide.

Example 10 QMP with Chromophore Detectable Label Moieties (FIGS.16(A)-(D))

QMP with chromophore detectable label moieties (FIGS. 16(A)-16(D)). Thetissue was deparaffinized and retrieved as described in the generalprocedures. Rabbit-anti-Ki-67 antibody incubation (37° C., 16 minutes)and washing were followed by secondary incubation with a goat polyclonalanti-rabbit, hapten labeled with nitropyrazole (NP) (37° C.; 8 minutes).After washing AP-conjugated mouse anti-NP monoclonal antibody was added(37° C.; 12 minutes). After incubating with the AP conjugate the slideswere washed with SSC. Monofluoro QMP-PEG8-Dabsyl (250 PM) (FIG. 16(A))or monofluoro QMP-TAMRA (250 μM) (FIG. 16(B)) or monofluoro QMP-Cy5 (250μM) (FIG. 16(C)) or monofluoro QMP-Rhodamine 110 (250 μM) (FIG. 16(D))were dissolved in 250 mM Tris, pH 10.0, 0.05% Brij-35. QMP turnover wasachieved by adding 100 μL of AP Enhancer followed by 100 μL theappropriate QMP and incubating at 37° C. for 32 minutes. The stainedtissue sections were counterstained with modified Mayer's hematoxylin(37° C.; 4 minutes) and then incubated with Bluing Reagent (37° C.; 4minutes). The slides were then dehydrated through a graded ethanolseries, cleared with xylene, and manually cover-slipped.

Examples 2-9 have demonstrated that QMP-reporters can be visualized byexisting detection chemistries (HRP/DAB & AP/Fast Red) and directly byfluorescence. This example demonstrates that if a chromophore with alarge extinction coefficient is the reporter molecule then the amplifiedQMP signal can be seen directly by brightfield microscopy. By selectingchromophores with different absorption wavelengths then a large range ofdiscrete colors can be created (FIGS. 16(A)-(D)). The ability to use asingle core molecule (QMP) with different reporters to generate multiplechromogenic detection systems is of fundamental importance for multiplexapplications. Previously to generate new chromogenic detection systems,new chemistries needed to be invented and each enzyme needed a differentapproach. The ability to use a single QMP core molecule with differentreporter molecules and/or different enzymatic recognition groupsprovides new tools for multiplex brightfield (and fluorescent) assays.

Example 11 Trapping Experiments

To probe the mechanism of staining and quenching, an attempt was made totrap the QM intermediate formed by monofluoro QM precursor 7 insolution-phase under similar conditions to those utilized in thestaining experiments (FIG. 26 ). QM precursor 7 was dissolved in pH 10Tris buffer followed by addition of a catalytic amount of AP. Thereaction progress was monitored by HPLC-MS (FIG. 27 , panels A-C).Within 10 minutes, complete conversion to the Tris adduct 7-tris wasobserved (panel C). In the absence of AP, no reaction took place,suggesting a high level of stability under the desired reactionconditions (panel B). These results strongly support the proposedmechanism of QM-mediated staining and elucidates Tris base as theprimary source of quenchers under the IHC staining conditions.

Additional trapping experiments were performed to demonstrate theability to generate and trap quinone methides in solution from QMprecursors comprising a phosphate, a galactoside and an acetate as theenzyme recognition group. Each QM precursor was treated in solution withthe corresponding enzyme, and the reaction progress was monitored byHPLC-MS (data not shown). Each substrate was diluted to 1 mM in a 250 mMTris solution at suitable pH for the cognate enzyme (10 for alkalinephosphatase, 8.0 for β-galactosidase, and 8.0 for lipase), followed byaddition of the cognate enzyme such that the final concentration ofenzyme in solution was 1 mM. The reactions were incubated at roomtemperature for 30 minutes, at which point the HPLC-MS was performed. Ineach case, the Tris-adduct was detected, demonstrating that the QM wasbeing successfully formed.

Example 12 Stability

One aspect of the present disclosure is that compounds with sufficientinstability are preferred in that the reaction should proceed quicklyupon interacting with the activating enzyme; however, this instabilitymust be balanced against the goal of making these compositions usefulwithin the scope of their intended use, as described herein. Inparticular, the use of these reagents as detection reagents forautomated IHC and ISH requires that the reagents be sufficiently stablein a container so that can be shipped to clinicians who can then storeand use the reagents over a significant period of time. Relevantshelf-lives for compositions of this type would be 12-month, 18-month,24-month, or greater stability. While storage conditions can bespecified, the use of the reagents on a clinical instrument oftenrequires that the reagent have significant room temperature stability.According to one embodiment, compositions of the present disclosure havesuitable stability for automated IHC and ISH. It should be noted thatsuitability for automated ISH and IHC does not require absolutestability; rather, that a substantial amount of the compound remainsafter an established amount of time so that the use of the reagent isnot adversely affected by the decomposition of the reagent.

Referring now to FIG. 28 , shown are four structures for compoundstested for suitable stability. Panel A is a para-di-substituted QMPconjugated to a TAMRA, panel B is a para-di-substituted QMP conjugatedto a Dabsyl, panel C is an ortho-di-substituted QMP conjugated to aDabsyl, and panel D is an ortho-di-substituted QMP conjugated to a Cy5.Referring now to Table 1, shown is the stability of compounds shown inFIG. 28 panels A and B in water at ambient temperature (20-25° C.). Thedegradation of the QMP dye conjugate appears to be independent of theidentity of the dye. While not limited to a particular theory ormechanism, it was understood that the main mode of degradation was thedisplacement of the fluoride by water to generate a compound that doesnot have the ability to stain tissue. Scheme 14 shows the hydrolyticdegradation of a representative QMP A to the non-staining compound B.

It was discovered that the compounds degrade too quickly to be stored inan un-buffered aqueous solution. In particular, it was observed thatonly about 42% of the QMP of FIG. 28 panel A and about 44% of the QMP ofFIG. 28 panel B remained after 12 days. The reagents were used on anautostainer and it was confirmed that the signal was diminished incomparison to a freshly synthesized reagent.

TABLE 1 Compound from FIG. 28 28(A) 28(B) 28(A) 28(A) 28(A) 28(B) 28(B)28(B) Solvent H₂O H₂O DMSO DMSO DMSO DMSO DMSO DMSO Temperature (° C.)20-25 20-25 30 45 60 30 45 60 Time 0.0 100.0  100.0  96.7 96.6 94.3 93.994.0 94.3 (Days) 0.2 88.5 90.6 — — — — — — 2.0 76.1 81.3 96.8 96.4 94.794.0 93.6 92.6 4.0 — — 96.4 96.2 93.4 94.2 93.8 91.6 5.0 68.8 73.8 — — —— — — 7.0 61.1 64.1 96.4 96.0 91.4 97.9 97.6 94.2 9.0 — — 96.3 95.9 89.598.0 97.3 92.4 11.0 — — 96.2 95.4 87.5 97.7 97.0 91.0 12.0 41.9 44.1 — —— — — — 14.0 — — 96.4 95.6 84.4 97.7 97.0 89.4 16.0 — — 96.5 95.0 83.597.8 97.0 87.8 18.0 — — 96.0 95.3 83.3 97.2 96.6 83.0 21.0 — — 95.7 94.577.9 93.6 92.8 80.4 23.0 — — 95.8 94.8 77.2 93.4 92.3 77.0 25.0 — — 95.694.5 76.5 93.3 91.6 75.9 28.0 — — 95.8 94.0 74.0 93.1 91.7 75.2To avoid the hydrolysis issue, one solution was to store the compoundsin an anhydrous, non-nucleophilic organic solvent (e.g. DMSO orpropylene carbonate). Table 1 shows data for an accelerated stabilitystudy where compounds of FIG. 28 panels A and B were stored at threeelevated temperatures (30, 45 and 60° C.). Again the behavior ofdifferent dye conjugates, when the QMP portion of the compound is keptthe same, is very similar. At elevated temperature a differentdegradation pathway was observed (Scheme 15). In particular, thedegradation of QMP in DMSO with trace water at elevated temperatures wasfound to hydrolyze at both the fluoro-group and the phosphate. Whenstored in DMSO, the QMP materials were diluted with a staining bufferclose to the time of use. It is understood that some autostainers arecapable of a pre-dilution step for preparing reagents; thus, thisapproach is provides the needed long term stability in a format whichcould operate within an intended use for the compounds.

To further investigate the aqueous stability of these compounds compoundfrom FIG. 28(A) was stored in three different buffers at various pH (seeTable 2). It was observed that lower pH's of the buffer solution extendthe shelf-life of the active staining compound.

TABLE 2 Compound from FIG. 28 28(B) 28(B) 28(B) Solvent Tris pH 10Acetate pH 4 Glycine pH 2 Temperature (° C.) 20-25 20-25 20-25 Time(Days) 0.0 100.0 100.0 100.0 0.1 69.8 97.2 — 0.2 55.1 95.0 99.6 1.0 11.189.9 93.7 1.2 7.6 88.5 94.4 1.9 2.5 86.3 93.0 2.0 — — — 2.1 1.9 85.691.3 4.0 — — — 5.0 1.3 73.4 87.2 7.0 — — — 8.0 1.3 64.8 82.6 9.0 1.362.5 82.2With an understanding of the degradation pathways and characteristics, asolvent system was designed for the purpose of extending shelf-life ofQMP compounds. It was discovered that a solvent system comprising a50/50 mixture of DMSO and 10 mM glycine buffer (pH 2.0), referred to asBuffer A herein, exhibits suitable stability. Table 3 shows data for anaccelerated stability study where compounds were stored at differenttemperatures (2, 25, 37 and 45 C) in a 50/50 mixture of DMSO and 10 mMglycine buffer (pH 2.0). At elevated temperature the degradation pathwaydescribed in FIG. 3C was still observed. However, at normal storageconditions (2-25° C.) the rate of decomposition of the QMP is reducedcompared to 100% aqueous systems.

TABLE 3 Compound from FIG. 28 28(A) 28(A) 28(A) 28(A) 28(C) 28(D)Solvent Buffer A Buffer A Buffer A Buffer A Buffer A Buffer ATemperature (° C.) 2 25 37 45 20-25 20-25 Time 0.0 96.3 96.3 94.3 94.2100.0 99.9 (Days) 1.0 — — — — 100.0 99.8 4.0 — — — — 100.0 95.4 8.0 96.294.1 76.9 80.1 100.0 95.6 10.0 — — — — 100.0 95.7 14.0 — — — — 100.095.1 15.0 96.2 93.3 64.9 68.4 — — 17.0 — — — — 100.0 95.8 22.0 96.7 92.455.2 59.1 — — 25.0 — — — —  97.8 95.5 30.0 96.7 91.2 46.4 49.9 — — 36.0— — — —  96.7 94.0 60.0 97.1 86.1 19.0 25.5 — —Table 3 also shows data for the stability of alternative QMP compounds(shown as FIG. 28 panels C and D in the same 50/50 mixture of DMSO and10 mM glycine buffer (pH 2.0). The initial data indicates that theortho-QMPs have an improved stability over the para-QMPs under thesestorage conditions.

Example 13—Salt and Cofactors

Magnesium is a required cofactor for alkaline phosphatase (AP) and thusit is expected the turnover rate of a QMP by AP would be dependent onthe concentration of magnesium. However, a surprising effect of theconcentration of magnesium salts on the staining quality was discovered.Again, it was understood that signal intensity would improve withincreasing concentration of magnesium because of its role as a cofactor.However, it was not expected that the quality of signal would improve.In particular, the qualities improved were signal localization,discreteness, and reduced diffusion. In fact, it was expected that thesequalities could actually be diminished by increasing magnesium as thegreater signal intensity could either bleed away or enhance the abilityto see bleeding away from the target location. Referring now to FIGS.29(A)-29(B), shown are photomicrographs of tonsil tissue stained for thepresence of CD8 with a QMP-TAMRA, with the singular variable of 0.125 Mmagnesium chloride (FIG. 29(B)) and 1.05 M magnesium chloride (FIG.29(A)). While photomicrographs and reproductions thereof do not show thesignificance of the difference as dramatically as one would wish, the1.05 M magnesium chloride sample exhibited much better stain qualitycompared to the 0.125 M magnesium sample. In illustrative embodiments,methods and compositions according to the present disclosure includegreater than about 0.1 M, 0.25 M, 0.5 M, 1.0 M or 1.25 M salt, such asmagnesium chloride. In other embodiments, the methods and compositionsaccording to the present disclosure include between about 0.1 M andabout 2 M, between about 0.25 M and 1.5 M, about 0.5 M to about 1.25 Mor about 1.0 M magnesium chloride. A similar effect was also observedfor NaCl concentration, but the effect was not as great.

This combined data, combined with the magnesium chloride concentrationstudy, led to the current kit configuration where there can be at leasttwo components; the first is a pH adjust (0.5 M Tris, pH 10.0) and thesecond is the QMP formulated in combination of organic solvent (e.g.DMSO) and 10 mM glycine (pH 2.0) with up to 1.0 M magnesium chloride.

Example 14 QMP with β-Galactosidase Trigger and Chromophore ReporterMoieties (FIGS. 30(A)-(C))

The tissue was deparaffinized and retrieved as described in the generalprocedures. Rabbit-anti-Ki-67 antibody incubation (37° C., 16 minutes)and washing were followed by secondary incubation with a goat polyclonalanti-rabbit, hapten labeled with biotin (37° C.; 8 minutes). Afterwashing, β-galactosidase (β-Gal) conjugated streptavidin (LifeTechnologies #S-931) was added (37° C.; 32 minutes). After incubatingwith the 1-Gal conjugate the slides were washed with SSC. Monofluoro(3-Gal-QMP-Cy5 (125 μM) (FIGS. 30(A)-30(B)) or β-Gal-QMP-Cy3 (100 PM)(FIG. 30(C)) were dissolved in 250 mM Tris, pH 8.0, 0.05% Brij-35. QMPturnover was achieved by adding 100 μL of AP Enhancer followed by 100 μLthe appropriate β-Gal-QMP and incubating at 37° C. for 32 minutes. Thestained tissue sections were counterstained with modified Mayer'shematoxylin (37° C.; 4 minutes) and then incubated with Bluing Reagent(37° C.; 4 minutes) (FIGS. 30(B)-30(C)) or Red Counterstain II (VMSI#780-2218) (37° C.; 4 minutes) (FIG. 30(A)). The slides were then rinsedwith a detergent water mixture, dehydrated through a graded ethanolseries, cleared with xylene, and manually cover-slipped.

β-Galactosidase has been as an enzyme for IHC detection previously with5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (BCIG) as the detectionreagent. However, it is the only color available, it is inconsistent,has poor sensitivity, and prone to fading (or washing out) during postprocessing of the slides. The examples in FIGS. 30(A)-30(C) show aβ-galactosidase based chromogenic IHC detection system that is notsusceptible to alcohol dehydration, has good sensitivity and can beeasily modified to generate a wide range of colors.

Example 15 Duplex Stain with Simultaneous Detection of AP and β-GalTriggered QMP (FIG. 31)

The tissue was deparaffinized and retrieved as described in the generalprocedures. Incubation with rabbit-anti-Ki-67 antibody andmouse-anti-Bcl-6 antibody (37° C., 16 minutes) and washing were followedby secondary antibody incubation with a goat polyclonal anti-rabbitantibody conjugated to biotin and a goat polyclonal anti-mouse antibodyconjugated to AP (37° C.; 12 minutes). The slides were subsequentlyincubated with β-Gal conjugated streptavidin (37° C.; 32 minutes)followed by washing with SSC. Phospho-QMP-Cy5 and β-Gal-QMP-Peg8-Dabsylwere dissolved in 250 mM Tris, pH 8.0, 0.05% Brij-35 to a finalconcentration of 250 μM each. QMP turnover was achieved by adding 100 μLof AP Enhancer followed by 100 μL of QMP mixture and incubating at 37°C. for 60 minutes. The stained tissue sections were counterstained withmodified Mayer's hematoxylin (37° C.; 4 minutes) and then incubated withBluing Reagent (37° C.; 4 minutes). They were then rinsed with adetergent water mixture, dehydrated through a graded ethanol series,cleared with xylene, and manually cover-slipped.

Some of the advantages of using multiple enzymes for multiplex assaysare the overall reduction in assay time and reduction of enzymeinactivation/elution steps. This example shows a dual chromogenic IHCdetection system where both enzymatic detections are carried out at thesame time. This result demonstrates the specificity of each QMP to itscognate enzyme and also the availability of sufficient binding sites forboth detections to occur unimpeded.

Example 16 Triplex Stain with Simultaneous Detection of HRP, AP andβ-Gal Substrates (FIGS. 32(A)-(B))

The tissue was deparaffinized and retrieved as described in the generalprocedures (FIGS. 32(A)-(B)). Rabbit-anti-PR (37° C., 16 minutes)incubation and washing were followed by secondary antibody incubationwith a goat polyclonal anti-rabbit antibody conjugated to biotin (37°C.; 8 minutes) and subsequently rabbit serum (37° C.; 8 minutes). Afterwashing, benzofuran (BF) labeled rabbit-anti-ER and dinitrophenol (DNP)labeled rabbit-anti-HER2 were simultaneously incubated (37° C.; 16minutes). The three enzyme conjugates: β-Gal conjugated streptavidin,HRP conjugated to mouse-anti-BF and AP conjugated to mouse-anti-NP; werethen applied (37° C.; 32 minutes) followed by washing with SSC.β-Gal-QMP-Cy5 and Phospho-QMP-PEG8-Dabsyl were dissolved in 250 mM Tris,pH 8.0, 0.05% Brij-35 to a final concentration of 300 μM each. Chromogenturnover was achieved by adding 100 μL of AP Enhancer followed by 100 μLof QMP mixture, 100 μL of DISCOVERY Purple (VMSI #760-229) and 100 μL ofH₂O₂ (0.01%) and incubating at 37° C. for 32 minutes. The stained tissuesections were counterstained with Hematoxylin II (37° C.; 4 minutes) andthen incubated with Bluing Reagent (37° C.; 4 minutes). They were thenrinsed with a detergent water mixture, dehydrated through a gradedethanol series, cleared with xylene, and manually cover-slipped.

This example shows a triple chromogenic IHC detection system where allenzymatic detections are carried out at the same time. This resultreinforces the specificity of each QMP to its cognate enzyme. It showsthe availability of sufficient binding sites for not only the QMPdetections to occur unimpeded, but also the fact that the HRP/tyramidedetection can still proceed.

Example 17 Quadruplex Stain with Sequential Detection of HRP (Twice), APand β-Gal Substrates (FIGS. 33-34)

The tissue was deparaffinized and retrieved as described in the generalprocedures. Rabbit-anti-PR (37° C., 16 minutes) incubation and washingwere followed by secondary antibody incubation with a goat polyclonalanti-rabbit antibody conjugated to biotin (37° C.; 8 minutes) andsubsequently rabbit serum (37° C.; 8 minutes). After washing,benzofurazan (BF) labeled rabbit-anti-ER, NP labeled rabbit anti-Ki67and dinitrophenol (DNP) labeled rabbit-anti-HER2 were simultaneouslyincubated (37° C.; 16 minutes). HRP conjugated to mouse-anti-DNP wasadded (37° C.; 8 minutes), and visualized via a brown precipitateproduced by HRP upon the addition of hydrogen peroxide and DAB (37° C.;8 minutes) which was further toned with copper sulfate. (3-Galconjugated to streptavidin, HRP conjugated to mouse-anti-BF and APconjugated to mouse-anti-NP were then applied (37° C.; 32 minutes). TheHRP was detected with 100 μL of DISCOVERY Purple (VMSI #760-229) and 100μL of H₂O₂ (0.01%) (37° C.; 32 minutes) followed by washing with SSC.β-Gal-QMP-Cy5 and Phospho-QMP-PEG8-Dabsyl were dissolved in 250 mM Tris,pH 8.0, 0.05% Brij-35 to a final concentration of 300 μM each. Chromogenturnover was achieved by adding 100 μL of AP Enhancer followed by 100 μLof QMP mixture, and incubating at 37° C. for 32 minutes. The stainedtissue sections were counterstained with Hematoxylin II (37° C.; 4minutes) and then incubated with Bluing Reagent (37° C.; 4 minutes).They were then rinsed with a detergent water mixture, dehydrated througha graded ethanol series, cleared with xylene, and manuallycover-slipped.

Example 18 Chromogenic ISH (FIGS. 35(A)-35(B))

The tissue was deparaffinized as described in the general proceduresfollowed by pretreatment with Cell Conditioning 2 (VMSI #950-123) (90°C.; 28 minutes) and treatment with Protease 3 (VMSI #780-4149) (37° C.;20 minutes). Chromosome 17 probe, DIG labeled (VMSI #760-1224) wasapplied to the tissue, denatured (80° C.; 20 minutes) and hybridized at44° C. for 6 hours. After three stringency washes at 76° C. with SSC,the sample was incubated with mouse-anti-DIG antibody (37° C.; 20minutes), followed by AP conjugated goat-anti-mouse antibody (37° C.; 24minutes). Phospho-QMP-PEG8-Dabsyl and phospho-QMP-Cy5 were dissolved in1:1 DMSO: 10 mM glycine buffer (pH 2.0) to a final concentration of 120μM QMP-Dabsyl and 30 μM QMP-Cy5 with 1 mM magnesium chloride. Afterwashing with SSC then 200 μL of pH adjust solution (500 mM Tris, pH10.0) and 100 uL of the QMP mixture were added (37° C.; 32 minutes). Thestained tissue sections were counterstained with Hematoxylin II (37° C.;4 minutes) and then incubated with Bluing Reagent (37° C.; 4 minutes).They were then rinsed with a detergent water mixture, dehydrated througha graded ethanol series, cleared with xylene, and manuallycover-slipped.

For ISH applications the ortho-QMP compounds showed better performancethan the para-QMP compounds. While the para-QMP compounds did generateISH signals, the intensity was low, the number of cells stained (cellcoverage) was inconsistent and the signal quality was not optimal. Theortho-QMP compounds generated stronger intensity signals, with increasedcell coverage and much better signal resolution with reduced diffusion.

Example 19 Quadruplex Stain with Sequential Detection of HRP (Twice) andAP (Twice) (FIG. 36)

FIG. 36 provides examples of four different staining protocols (A-D)showing the same biomarkers (panel A—Her2 (membrane), panel B—Ki-67(nuclear), panel C—ER (nuclear) and panel D—PR (nuclear)) on FFPE breasttissue with interchanged and/or different color combinations (40×magnification). As there were three nuclear markers and one membranemarker, it was possible to use DAB for the Her2 membrane stain in someprotocols, avoiding the overlap of DAB with other colors. Mixes of thenuclear detections (yellow, blue & purple) generated different colorcombinations depending on intensity.

The assay included sequential detection of the biomarkers using two HRPbased detections and two AP QMP based detections. This demonstrated theflexibility and interchangeability of the disclosed detection systems.

Example 20 Quadruplex Stain with Sequential Detection of HRP (Twice) andAP (Twice) (FIG. 37)

FIG. 37 provides examples of different staining protocols (panels A-B orpanels C-D) showing the same biomarkers (panel A—CD3 (membrane), panelB—CD8 (membrane), panel C—CD20 (membrane) (or CD68 (membrane)) and panelD—FoxP3 (nuclear) on FFPE tonsil tissue) with interchanged and/ordifferent color combinations (5× magnification). Sequential detectionwas utilized, using two HRP based detections and two AP QMP baseddetections, further demonstrating the flexibility and interchangeabilityof the disclosed detection systems.

Example 21

Following the protocol outlined in Example 10, breast tissue was stainedwith a mouse-anti-E-cadherin monoclonal primary antibody (Ventana#790-4497) using either compound 36 at a concentration of 500 μM (FIG.38(A)) or compound 28 at a concentration of 400 μM (FIG. 38(B)). It wasexpected that both would give equivalent results, but surprisinglycompound 28 (the ortho-QMP) showed better performance than compound 36(the para-QMP). The staining quality (signal localization anddiscreteness) was the same for both compounds, but the stainingintensity was higher for compound 28, even using 20% lower concentrationthan compound 36.

The ortho-QMP compounds offer significant improvement to IHC stainingperformance, ISH staining performance and improved aqueous stabilitycompared to any of the compounds described previously.

In view of the many possible embodiments to which the principles of thedisclosed invention may be applied, it should be recognized that theillustrated embodiments are only preferred examples of the invention andshould not be taken as limiting the scope of the invention. Rather, thescope of the invention is defined by the following claims. We thereforeclaim as our invention all that comes within the scope and spirit ofthese claims.

ADDITIONAL EXEMPLARY EMBODIMENTS

The following additional embodiments are also specifically disclosed.This is not an exhaustive list.

1. A compound, having a formula

or a salt or solvate thereof, wherein:

-   -   Z is O, S or NR^(a) and R¹ is an enzyme recognition group, or        ZR¹ is an enzyme recognition group;    -   R⁸ is —C(LG)(R⁵)(R³R⁴), —R³R⁴ or —C(LG)(R⁵)(R⁶);    -   R⁹, R¹¹ and R¹² are each independently hydrogen, halo, cyano,        aliphatic, alkoxy, NO₂, N(R^(c))₂, aryl, haloalkyl, —C(O)alkyl,        —C(S)alkyl, —C(O)OH, —C(O)Oalkyl, —C(O)NHR^(c), —C(O)N(R^(c))₂,        —R³R⁴ or two adjacent groups together form an aliphatic ring or        aryl ring;    -   R¹⁰ is hydrogen, halo, cyano, aliphatic, alkoxy, NO₂, N(R^(c))₂,        aryl, haloalkyl, —C(O)alkyl, —C(S)alkyl, —C(O)OH, —C(O)Oalkyl,        —C(O)NHR^(c), —C(O)N(R^(c))₂, —R³R⁴, —C(LG)(R⁵)(R⁶) or with one        of R⁹ or R¹¹ form an aliphatic ring or aryl ring;    -   LG is a leaving group, or ZR¹ and LG together form a        phosphodiester;    -   R³ is a linker or a bond;    -   R⁴ is a detectable label;    -   each R⁵ is independently hydrogen, halo, cyano, lower alkyl,        lower haloalkyl, —C(O)alkyl, —C(S)alkyl, —C(O)OH, —C(O)Oalkyl,        —C(O)NHR^(c) or —C(O)N(R^(c))₂;    -   each R⁶ is independently hydrogen, halo, cyano, lower alkyl,        lower haloalkyl, —C(O)alkyl, —C(S)alkyl, —C(O)OH, —C(O)Oalkyl,        —C(O)NHR^(c) or —C(O)N(R^(c))₂;    -   R^(a) is hydrogen or aliphatic;    -   each R^(c) independently is hydrogen, aryl, aliphatic or        heteroaliphatic, or two R^(c) moieties together form a        heteroaliphatic ring; and    -   at least one of R⁸ and R¹⁰ comprises LG, and at least one of R⁸        and R¹⁰ comprises R³R⁴; and    -   if LG is halo, then R⁵ and R⁶ are not halo.

2. The compound of embodiment 1, wherein the compound has a formula

3. The compound of embodiment 1, wherein the compound has a formulaselected from

4. The compound of embodiment 1, wherein R¹ or ZR¹ is a phosphate,amide, nitro, urea, sulfate, methyl, ester, beta-lactam, sugar, or LGand ZR¹ together form a phosphodiester.

5. The compound of embodiment 4, wherein Z is O.

6. The compound of embodiment 4, wherein ZR¹ is —OP(O)(OH)₂, NO₂,—NHC(O)R, —OC(O)CH₃, —OC(O)CH₂CH₃, —NHC(O)NH₂, —OS(O)₂OH, OCH₃, or asalt thereof.

7. The compound of embodiment 4, wherein the sugar is α-glucose,β-glucose, α-galactoside, β-galactoside, α-glucuronose or β-glucuronose.

8. The compound of embodiment 4, wherein the beta-lactam is

-   -   R is alkyl; and    -   Z is O or S.

9. The compound of embodiment 1, wherein LG is halide, sulfate ester,carboxylate, inorganic ester, thiolate, amine, aryloxy, alkoxy, orheteroaryl.

10. The compound of embodiment 1, wherein:

-   -   LG is fluoride, chloride, azide, acetate, methoxy, ethoxy,        isopropoxy, phenoxide, —OS(O)₂CH₃, —OS(O)₂C₆H₄CH₃, —OS(O)₂C₆H₅,        —OS(O)₂C₆H₄CX₃, —OC₆H₅, —N₂ ⁺, —NH₃ ⁺, —NC₆H₅ ⁺, —O-alkyl,        —OC(O)alkyl, —OC(O)H, —N(R^(b))₃ ⁺ or        1,4-diazabicyclo[2.2.2]octane;    -   each X independently is fluoro, chloro, bromo or iodo; and    -   each R^(b) independently is hydrogen or lower alkyl, or two        R^(b) moieties together form a heteroaliphatic ring.

11. The compound of embodiment 10, wherein LG is F.

12. The compound of embodiment 1, wherein R³ is —(CH₂)_(n)NH—,—O(CH₂)_(n)NH—, —N(H)C(O)(CH₂)_(n)NH—, —C(O)N(H)(CH₂)_(n)NH—,—(CH₂)_(n)O—, —O(CH₂)_(n)O—, —O(CH₂CH₂O)_(n)—, —N(H)C(O)(CH₂)_(n)O—,—C(O)N(H)(CH₂)_(n)O—, —C(O)N(H)(CH₂CH₂O)_(n)—, —(CH₂)_(n)S—,—O(CH₂)_(n)S—, —N(H)C(O)(CH₂)_(n)S—, —C(O)N(H)(CH₂)_(n)S—,—(CH₂)_(n)NH—, —C(O)N(H)(CH₂CH₂O)_(n)CH₂CH₂NH,—C(O)(CH₂CH₂O)_(n)CH₂CH₂NH—, —C(O)N(H)(CH₂)_(n)NHC(O)CH(CH₃)(CH₂)_(n)NH—or —N(H)(CH₂)_(n)NH—, where each n independently is 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11 or 12.

13. The compound of embodiment 1, wherein R³ is —CH₂CH₂NH—, —OCH₂CH₂NH—,—NHCO(CH₂)₅NH—, —CONH(CH₂)₅NH—, —NHCO(CH₂)₆NH—, —CONH(CH₂)₆NH—,—CONH(CH₂)₂NH—, —(CH₂CH₂O)₄—, —(CH₂CH₂O)₈—,—C(O)N(H)(CH₂CH₂O)₂CH₂CH₂NH—, —CO(CH₂CH₂O)₄CH₂CH₂NH—,—CO(CH₂CH₂O)₈CH₂CH₂NH—, —C(O)N(H)(CH₂)₆NHC(O)CH(CH₃)(CH₂)₄NH—,

14. The compound of embodiment 1, wherein R⁴ is a chromogen, afluorophore, a luminophore, a hapten or a combination thereof.

15. The compound of embodiment 14, wherein R⁴ is

16. The compound of embodiment 1, wherein the compound is

and

-   -   R⁴ is a chromogen, a fluorophore, a luminophore, or a hapten.

17. The compound of embodiment 1, wherein the compound is

and

-   -   R⁴ is a chromogen, a fluorophore, a luminophore, or a hapten.

18. The compound of embodiment 16, wherein R⁴ is

19. A method of detecting a first target in a biological sample,comprising:

-   -   contacting the biological sample with a first detection probe        specific to the first target;    -   contacting the biological sample with a first labeling        conjugate, comprising a first enzyme;    -   contacting the biological sample with a first quinone methide        analog precursor comprising a first enzyme recognition group and        a first detectable label, wherein the first enzyme cleaves the        first enzyme recognition group, thereby converting the first        quinone methide analog precursor into a first reactive quinone        methide analog which covalently binds to the biological sample        proximally to or directly on the first target, contacting the        biological sample comprising        -   (i) contacting the biological sample with the first quinone            methide analog precursor at a precursor concentration            effective to give a desired level of amplification;        -   (ii) contacting the biological sample with the first quinone            methide analog precursor at a pH effective to reduce            diffusion and/or off-target staining to a desired amount;        -   (iii) contacting the biological sample with the first            quinone methide analog precursor in the presence of a salt            at a salt concentration effective to reduce diffusion and/or            off-target staining to a desired amount;        -   (iv) contacting the biological sample with a compound            according to claim 1; or        -   (v) any combination thereof; and    -   detecting the first target by detecting the first detectable        label.

20. The method of embodiment 19, wherein contacting the biologicalsample comprises contacting the biological sample with a compoundaccording to claim 1.

21. The method of embodiment 19, wherein the salt concentration is from0.1 M to 2 M.

22. The method of embodiment 21, wherein the salt concentration is from0.5 M to 1.25 M.

23. The method of embodiment 19, wherein the salt is magnesium chloride.

24. The method of embodiment 19, comprising contacting the biologicalsample with a first quinone methide analog precursor at a pH effectiveto reduce diffusion and/or off-target staining to a desired amount.

25. The method of embodiment 24, wherein the pH is from greater than 7to 14.

26. The method of embodiment 25, wherein the pH is from 8 to 12.

27. The method of embodiment 19, comprising contacting the biologicalsample with a first quinone methide analog precursor at a precursorconcentration effective to give a desired level of amplification.

28. The method of embodiment 27, wherein the precursor concentration isfrom greater than zero to 1 mM.

29. The method of embodiment 28, wherein the precursor concentration isfrom 50 nM to 100 μM 30. The method of embodiment 29, wherein theprecursor concentration is from 100 nM to 1 μM.

31. The method of embodiment 19, wherein the biological sample comprisesformalin-fixed, paraffin-embedded tissue.

32. The method of embodiment 19, wherein the method is an automatedprocess.

33. The method of embodiment 19 wherein the first detection probecomprises an oligonucleotide, an antibody, or an antibody fragment.

34. The method of embodiment 33, wherein the first detection probecomprises a hapten-labeled oligonucleotide.

35. The method of embodiment 19, wherein the first detection probecomprises an oxazole hapten, pyrazole hapten, thiazole hapten, nitroarylhapten, benzofuran hapten, triterpene hapten, urea hapten, thioureahapten, rotenoid hapten, coumarin hapten, cyclolignan hapten,di-nitrophenyl hapten, biotin hapten, digoxigenin hapten, fluoresceinhapten, or rhodamine hapten.

36. The method of embodiment 19, wherein the first labeling conjugatecomprises an antibody coupled to the first enzyme.

37. The method of embodiment 36, wherein the antibody is an anti-speciesor an anti-hapten antibody.

38. The method of embodiment 19, wherein the first labeling conjugate isassociated with the first detection probe.

39. The method of embodiment 38, wherein the first labeling conjugate isdirectly associated with the first detection probe.

40. The method of embodiment 38, wherein the first labeling conjugate isindirectly associated with the first detection probe.

41. The method of embodiment 36, wherein the first detection probecomprises a first anti-species antibody and the first labeling probecomprises a second anti-species antibody.

42. The method of embodiment 36, wherein the first detection probecomprises a hapten and the first labeling probe comprises an anti-haptenantibody.

43. The method of embodiment 19, wherein the first enzyme is aphosphatase, phosphodiesterase, esterase, lipase, amidase, protease,nitroreductase, urease, sulfatase, cytochrome P450, alpha-glucosidase,beta-glucosidase, beta-lactamase, alpha-glucoronidase,beta-glucoronidase, alpha-galactosidase, beta-galactosidase,alpha-lactase or beta-lactase.

44. The method of embodiment 19, wherein the first enzyme recognitiongroup is a phosphate, phosphodiester, amide, nitro, urea, sulfate,methyl, ester, alpha-glucose, beta-glucose, beta-lactam,alpha-galactoside, beta-galactoside, alpha-lactose, beta-lactose,alpha-glucuronoside or beta-glucuronoside.

45. The method of embodiment 19, wherein the first reactive quinonemethide analog reacts with a nucleophilic site within the biologicalsample, the first labeling conjugate, the first detection probe, orcombinations thereof.

46. The method of embodiment 45, wherein the nucleophilic site comprisesan amino, sulfhydryl, or hydroxyl group on an amino acid or nucleic acidresidue.

47. The method of embodiment 19, wherein the first quinone methideanalog precursor has a formula

or a salt or solvate thereof;

-   -   Z is O, S or NR^(a) and R¹ is an enzyme recognition group, or        ZR¹ is an enzyme recognition group;    -   R⁸ is —C(LG)(R⁵)(R³R⁴), —R³R⁴ or —C(LG)(R⁵)(R⁶);    -   R⁹, R¹¹ and R¹² are each independently hydrogen, halo, cyano,        aliphatic, alkoxy, NO₂, N(R^(c))₂, aryl, haloalkyl, —C(O)alkyl,        —C(S)alkyl, —C(O)OH, —C(O)Oalkyl, —C(O)NHR^(c), —C(O)N(R^(c))₂,        —R³R⁴ or two adjacent groups together form an aliphatic ring or        aryl ring;    -   R¹⁰ is hydrogen, halo, cyano, aliphatic, alkoxy, NO₂, N(R^(c))₂,        aryl, haloalkyl, —C(O)alkyl, —C(S)alkyl, —C(O)OH, —C(O)Oalkyl,        —C(O)NHR^(c), —C(O)N(R^(c))₂, —R³R⁴, —C(LG)(R⁵)(R⁶) or with one        of R⁹ or R¹¹ form an aliphatic ring or aryl ring;    -   LG is a leaving group, or ZR¹ and LG together form a        phosphodiester;    -   R³ is a bond or a linker;    -   R⁴ is a detectable label;    -   each R^(a) independently is hydrogen or aliphatic;    -   each R^(c) independently is hydrogen, aryl, aliphatic or        heteroaliphatic, or two R^(c) moieties together form a        heteroaliphatic ring;    -   each R⁵ is independently hydrogen, halo, cyano, lower alkyl,        lower haloalkyl, —C(O)alkyl, —C(S)alkyl, —C(O)OH, —C(O)Oalkyl,        —C(O)NHR^(c) or —C(O)N(R^(c))₂;    -   each R⁶ is independently hydrogen, halo, cyano, lower alkyl,        lower haloalkyl, —C(O)alkyl, —C(S)alkyl, —C(O)OH, —C(O)Oalkyl,        —C(O)NHR^(c) or —C(O)N(R^(c))₂;    -   at least one of R⁸ and R¹⁰ comprises LG;    -   at least one of R⁸-R¹² comprises R³R⁴; and    -   if LG is halo, then R⁵ and R⁶ are not halo.

48. The method of embodiment 47, wherein the first quinone methideprecursor has a formula

49. The method of embodiment 48, wherein the first quinone methideanalog precursor has a structure selected from

wherein R⁴ is a chromogen, a fluorophore, a luminophore, or a hapten.

50. The method of embodiment 47, wherein the first quinone methideanalog precursor has a formula selected from

51. The method of embodiment 50, wherein the first quinone methideanalog precursor has a structure selected from

wherein R⁴ is a chromogen, a fluorophore, a luminophore, or a hapten.

52. The method of embodiment 19, wherein the first quinone methideanalog precursor has a structure selected from

or a salt or solvate thereof;

-   -   Z is O, S or NR^(a) and R¹ is an enzyme recognition group, or        ZR¹ is an enzyme recognition group;    -   R⁹ and R¹¹-R²⁰ are each independently hydrogen, halo, cyano,        aliphatic, alkoxy, NO₂, N(R^(c))₂, aryl, haloalkyl, —C(O)alkyl,        —C(S)alkyl, —C(O)OH, —C(O)Oalkyl, —C(O)NHR^(c), —C(O)N(R^(c))₂,        —R³R⁴ or two adjacent groups together form an aliphatic ring or        aryl ring;    -   R¹⁰ is hydrogen, halo, cyano, aliphatic, alkoxy, NO₂, N(R^(c))₂,        aryl, haloalkyl, —C(O)alkyl, —C(S)alkyl, —C(O)OH, —C(O)Oalkyl,        —C(O)NHR^(c), —C(O)N(R^(c))₂, —R³R⁴, —C(LG)(R⁵)(R⁶) or with one        of R⁹ or R¹¹ form an aliphatic ring or aryl ring;    -   LG is a leaving group, or ZR¹ and LG together form a        phosphodiester;    -   R³ is a bond or a linker;    -   R⁴ is a detectable label;    -   each R^(a) independently is hydrogen or aliphatic;    -   each R^(c) independently is hydrogen, aryl, aliphatic or        heteroaliphatic, or two R^(c) moieties together form a        heteroaliphatic ring;    -   each R⁵ is independently hydrogen, halo, cyano, lower alkyl,        lower haloalkyl, —C(O)alkyl, —C(S)alkyl, —C(O)OH, —C(O)Oalkyl,        —C(O)NHR^(c) or —C(O)N(R^(c))₂;    -   each R⁶ is independently hydrogen, halo, cyano, lower alkyl,        lower haloalkyl, —C(O)alkyl, —C(S)alkyl, —C(O)OH, —C(O)Oalkyl,        —C(O)NHR^(c) or —C(O)N(R^(c))₂;    -   at least one of R⁸ and R¹⁰ comprises LG;    -   at least one of R⁹-R²⁰ comprises R³R⁴; and    -   if LG is halo, then R⁵ and R⁶ are not halo.

53. The method of embodiment 19, wherein the first quinone methideanalog precursor has a formula selected from

-   -   Z is O, S or NR^(a) and R¹ is an enzyme recognition group, or        ZR¹ is an enzyme recognition group;    -   R⁵ and R⁶ are each independently hydrogen, halo, cyano, lower        alkyl, lower haloalkyl, —C(O)alkyl, —C(S)alkyl, —C(O)OH,        —C(O)Oalkyl, —C(O)NHR^(c) or —C(O)N(R^(c))₂;    -   R²⁵-R²⁹ are each independently hydrogen, halo, cyano, aliphatic,        alkoxy, NO₂, N(R^(c))₂, aryl, haloalkyl, —C(O)alkyl, —C(S)alkyl,        —C(O)OH, —C(O)Oalkyl, —C(O)NHR^(c), —C(O)N(R^(c))₂, —R³R⁴, or        two adjacent groups together form an aliphatic ring or aryl        ring; each R^(c) independently is hydrogen, aryl, aliphatic or        heteroaliphatic, or two R^(c) moieties together form a        heteroaliphatic ring;    -   LG is a leaving group, or ZR¹ and LG together form a        phosphodiester;    -   R³ is a bond or a linker;    -   R⁴ is a detectable label;    -   each R^(a) independently is hydrogen or aliphatic;    -   each R^(c) independently is hydrogen, aryl, aliphatic or        heteroaliphatic, or two R^(c) moieties together form a        heteroaliphatic ring;    -   at least one of R⁵, R⁶ and R²⁵-R²⁹ comprises R³R⁴; and    -   when LG is halo, R⁵ and R⁶ are not halo.

54. The method of embodiment 19, wherein the first quinone methideanalog precursor has a formula

or a salt or solvate thereof;

-   -   Z is O, S or NR^(a) and R¹ is an enzyme recognition group, or        ZR¹ is an enzyme recognition group;    -   LG is a leaving group;    -   R³ is a bond or a linker;    -   R⁴ is a detectable label;    -   R²¹, R²², R²³ and R²⁴ are each independently hydrogen, halo,        cyano, aliphatic, alkoxy, NO₂, N(R^(c))₂, aryl, haloalkyl,        —C(O)alkyl, —C(S)alkyl, —C(O)OH, —C(O)Oalkyl, —C(O)NHR^(c),        —C(O)N(R^(c))₂, —R³R⁴, or two adjacent groups together form an        aliphatic ring or aryl ring;    -   each R^(a) independently is hydrogen or aliphatic; and    -   each R^(c) independently is hydrogen, aryl, aliphatic or        heteroaliphatic, or two R^(c) moieties together form a        heteroaliphatic ring.

55. The method of embodiment 54, wherein the first quinone methideanalog precursor is selected from

where R⁴ is selected from a hapten, fluorophore, luminophore, orchromogen.

56. The method of embodiment 19, wherein the target is a peptide,polypeptide, protein or nucleic acid sequence.

57. The method of embodiment 19, wherein the method is a multiplexedmethod.

58. The method of embodiment 57, further comprising:

-   -   contacting the biological sample with a second binding moiety        specific to a second target;    -   labeling the second target with a second enzyme through the        second binding moiety;    -   contacting the biological sample with a second detection        precursor compound, the second detection precursor compound        interacting with the second enzyme to deposit a second detection        compound directly on or proximally to the second target; and        detecting the second detection compound.

59. The method of embodiment 58, wherein the first enzyme and secondenzyme are different enzymes.

60. The method of embodiment 58, wherein the first enzyme reactsselectively with the first quinone methide analog precursor, and thesecond enzyme reacts selectively with the second detection precursorcompound.

61. The method of embodiment 58, where the method does not comprise anenzyme deactivation step.

62. The method of embodiment 58, wherein the first enzyme is aphosphatase, phosphodiesterase, esterase, lipase, amidase, protease,nitroreductase, urease, sulfatase, cytochrome P450, alpha-glucosidase,beta-glucosidase, beta-lactamase, alpha-glucoronidase,beta-glucoronidase, alpha-galactosidase, beta-galactosidase,alpha-lactase or beta-lactase.

63. The method of embodiment 58, wherein the first enzyme is an alkalinephosphatase and the first enzyme recognition group is a phosphate.

64. The method of embodiment 58, wherein the second enzyme is aperoxidase.

65. The method of embodiment 58 wherein the second detection precursorcompound is a second quinone methide analog precursor comprising asecond enzyme recognition group and a second detectable label, thesecond quinone methide analog precursor interacting with the secondenzyme to form a second quinone methide analog which covalently binds tothe biological sample proximally to or directly on the second target,and wherein detecting the second detection compound comprises detectingthe second detectable label.

66. The method of embodiment 58, wherein the second enzyme is a3-galactosidase and the second enzyme recognition group is aβ-galactoside.

67. The method of embodiment 58, wherein contacting the tissue with thefirst binding moiety specific to the first target and contacting thetissue with the second binding moiety specific to the second targetoccur substantially contemporaneously.

68. The method of embodiment 58, wherein labeling the first target withthe first enzyme through the first binding moiety and labeling thesecond target with the second enzyme through the second binding moietyoccur substantially contemporaneously.

69. The method of embodiment 58, wherein contacting the tissue with thefirst binding moiety specific to the first target and contacting thetissue with the second binding moiety specific to the second targetoccur sequentially.

70. The method of embodiment 58, wherein labeling the first target withthe first enzyme through the first binding moiety and labeling thesecond target with the second enzyme through the second binding moietyoccur sequentially.

71. The method of embodiment 58, further comprising:

-   -   contacting the biological sample with a third binding moiety        specific to a third distinct target;    -   labeling the third target with a third enzyme through the third        binding moiety;    -   contacting the biological sample with a third detection        precursor compound, the third detection precursor compound        interacting with the third enzyme to deposit a third detection        compound directly on or proximally to the third target; and    -   detecting the third detection compound.

72. The method of embodiment 71, wherein the first enzyme is an alkalinephosphatase and the first enzyme recognition group is a phosphate, thesecond enzyme is a β-galactosidase and second detection precursorcompound is a second quinone methide analog precursor comprising aβ-galactoside, and the third enzyme is a peroxidase.

73. A kit, comprising:

-   -   an enzyme-antibody conjugate;    -   a quinone methide analog precursor;    -   a solvent mixture; and    -   a pH adjust solution.

74. The kit of embodiment 73, wherein the solvent mixture comprises anorganic solvent and an aqueous buffer.

75. The kit of embodiment 74, wherein the organic solvent is DMSO.

76. The kit of embodiment 74, wherein the aqueous buffer has a pH rangeof from pH 0 to pH 5.

77. The kit of embodiment 76, wherein the pH range is from pH 1 to pH 3.

78. The kit of embodiment 73, wherein the pH adjust solution has a pHrange of from pH 8 to pH 12.

79. The kit of embodiment 73, further comprising magnesium chloride at aconcentration of from 0.25 M to 1.5 M.

80. The kit of embodiment 73, wherein the quinone methide analogprecursor is a compound according to embodiment 1.

81. The kit of embodiment 73, wherein:

-   -   the quinone methide analog precursor is a compound according to        claim 1;    -   the solvent mixture comprises DMSO and a glycine buffer at pH 2;    -   the pH adjust solution is a Tris buffer with a pH range of from        pH 8 to pH 10; and    -   further comprising magnesium chloride at a concentration of from        0.5M to 1.25M.

1. A compound having Formula (III):

wherein each Q is carbon; each R⁷ is independently selected fromhydrogen, —Z—R¹, a moiety comprising a LG group, a moiety comprising adetectable label, a moiety comprising a LG group and a detectable label,a halo group, an aliphatic group, an alkoxy group, or where two adjacentR⁷ groups together form an aliphatic ring or aryl ring where the formedaliphatic ring or aryl ring may be substituted with at least one of amoiety comprising LG group and a moiety comprising a detectable label;—Z—R¹ is —OP(O)(OH)₂, —SP(O)(OH)₂, —O-sugar or a derivative or analogthereof, —NR^(a)P(O)(OH)₂, —OC(═O)R^(a), —N(R^(a))C(═O)R^(a), —NO₂,—NR^(a)—C(═O)—N(R^(c))₂, —OSO₃H, —OR^(a), or a β-lactam-containingmoiety; LG is a halide, alkoxy, carboxylate, inorganic ester, thiolate,amine, carboxylate, azide, sulfate ester, aryloxy, phenoxide or—N(R^(b))₃ ⁺ where each R^(b) is independently hydrogen or a lower alkylor two R^(b) moieties together form a heteroaliphatic ring; m is 1, atleast one of the R⁷ groups comprises —Z—R¹; and the detectable label isselected from the group consisting of a hapten, a chromophore, a dye,and a fluorophore; and wherein the compound of Formula (III) comprisesat least one detectable label.
 2. The compound of claim 1, wherein —Z—R¹is —O-sugar or a derivative or analog thereof.
 3. The compound of claim2, wherein —O-sugar is —O-alpha-glucose, —O-beta-glucose,—O-alpha-galactose, —O-beta-galactose, —O-alpha-lactose,—O-beta-lactose, —O-alpha-glucuronic acid, and —O-beta-glucuronic acid.4. The compound of claim 1, wherein the β-lactam-containing moiety is

wherein R is alkyl; and Z is O or S.
 5. A quinone methide analogprecursor compound having Formula (IV) or a salt or solvate thereof:

wherein R⁸ is —C(LG)(R⁵)(R³R⁴), —R³R⁴ or —C(LG)(R⁵)(R⁶); R⁹, R¹¹ and R¹²are each independently hydrogen, halo, cyano, aliphatic, alkoxy, NO₂,N(R^(c))₂, aryl, haloalkyl, —C(O)alkyl, —C(S)alkyl, —C(O)OH,—C(O)Oalkyl, —C(O)NHR^(c), —C(O)N(R^(c))₂, —R³R⁴, or where R^(u) and R¹²together form an aliphatic ring or aryl ring; R¹⁰ is hydrogen, halo,cyano, aliphatic, alkoxy, NO₂, N(R^(c))₂, aryl, haloalkyl, —C(O)alkyl,—C(S)alkyl, —C(O)OH, —C(O)Oalkyl, —C(O)NHR^(c), —C(O)N(R^(c))₂—R³R⁴,—C(LG)(R⁵)(R⁶), or where R¹⁰ together with one of R⁹ or R¹¹ form analiphatic ring or aryl ring; LG is a leaving group; Z—R¹ is —OP(O)(OH)₂,—SP(O)(OH)₂, —NR^(a)P(O)(OH)₂, —OC(═O)R^(a), —N(R^(a))C(═O)R^(a), —NO₂,—NR^(a)—C(═O)—N(R^(c))₂, —OSO₃H, —OR^(a), —O-β-lactam-containing moiety,—S-β-lactam-containing moiety, or —O-enzyme-appropriate sugar; R³ is abond, —(CH₂)_(n)NH—, —O(CH₂)_(n)NH—, —N(H)C(O)(CH₂)_(n)NH—,—C(O)N(H)(CH₂)_(n)NH—, —(CH₂)_(n)O—, —O(CH₂)_(n)O—, —O(CH₂CH₂O)_(n)—,—N(H)C(O)(CH₂)_(n)O—, —C(O)N(H)(CH₂)_(n)O—, —C(O)N(H)(CH₂CH₂O)_(n)—,—(CH₂)_(n)S—, —O(CH₂)_(n)S—, —N(H)C(O)(CH₂)_(n)S—, —C(O)N(H)(CH₂)_(n)S—,—(CH₂)_(n)NH—, —C(O)N(H)(CH₂CH₂O)_(n)CH₂CH₂NH,—C(O)(CH₂CH₂O)_(n)CH₂CH₂NH—, —C(O)N(H)(CH₂)_(n)NHC(O)CH(CH₃)(CH₂)_(n)NH—or —N(H)(CH₂)_(n)NH—,

 where each n is independently an integer ranging from 1 to 12; R⁴ is achromogen, a fluorophore, a luminophore, a hapten or a combinationthereof; each R⁵ is independently hydrogen, halo, cyano, lower alkyl,lower haloalkyl, —C(O)alkyl, —C(S)alkyl, —C(O)OH, —C(O)Oalkyl,—C(O)NHR^(c) or —C(O)N(R)₂; each R⁶ is independently hydrogen, halo,cyano, lower alkyl, lower haloalkyl, —C(O)alkyl, —C(S)alkyl, —C(O)OH,—C(O)Oalkyl, —C(O)NHR^(c) or —C(O)N(R)₂; each R^(a) is hydrogen; eachR^(c) independently is hydrogen, aryl, aliphatic or heteroaliphatic, ortwo R^(c) moieties together form a heteroaliphatic ring; and at leastone of R⁸ and R¹⁰ comprises LG, and at least one of R⁸ and R¹⁰ comprisesR³R⁴; and if LG is halo, then R⁵ and R⁶ are not halo.
 6. The quinonemethide analog precursor compound of claim 5, wherein the quinonemethide analog precursor compound has a Formula:


7. The quinone methide analog precursor compound of claim 5, wherein thequinone methide analog precursor compound has a Formula:


8. The quinone methide analog precursor compound of claim 5, wherein thequinone methide analog precursor compound has a Formula:

where R¹³-R²⁰ are each independently hydrogen, halo, cyano, aliphatic,alkoxy, —NO₂, —N(R^(c))₂, aryl, haloalkyl, —C(O)alkyl, —C(S)alkyl,—C(O)OH, —C(O)Oalkyl, —C(O)NHR^(c), —C(O)N(R^(c))₂, —R³R⁴ or twoadjacent groups together form an aliphatic ring or aryl ring, and atleast one of R¹-R²⁰ comprises —R³R⁴.
 9. The quinone methide analogprecursor compound of claim 5, wherein Z—R¹ is —OP(O)(OH)₂, —NO₂,—NHC(O)R, —OC(O)CH₃, —OC(O)CH₂CH₃, —NHC(O)NH₂, —OS(O)₂OH, OCH₃, or asalt thereof.
 10. The quinone methide analog precursor compound of claim5, wherein the sugar is α-glucose, β-glucose, α-galactoside,β-galactoside, α-glucuronose or β-glucuronose.
 11. The quinone methideanalog precursor compound of claim 5, wherein LG is fluoride, chloride,azide, acetate, methoxy, ethoxy, isopropoxy, phenoxide, —OS(O)₂CH₃,—OS(O)₂C₆H₄CH₃, —OS(O)₂C₆H₅, —OS(O)₂C₆H₄CX₃, —OC₆H₅, —N₂ ⁺, —NH₃ ⁺,—NC₆H₅ ⁺, —O-alkyl, —OC(O)alkyl, —OC(O)H, —N(R^(b))₃ ⁺, or1,4-diazabicyclo[2.2.2]octane; wherein each X is independently isfluoro, chloro, bromo or iodo; and wherein each R^(b) independently ishydrogen or lower alkyl, or two R^(b) moieties together form aheteroaliphatic ring.
 12. The quinone methide analog precursor compoundof claim 5, wherein R⁴ is


13. The quinone methide analog precursor compound of claim 5, whereinthe quinone methide analog precursor compound is selected from the groupconsisting of:


14. The quinone methide analog precursor compound of claim 5, whereinthe quinone methide analog precursor compound is selected from the groupconsisting of


15. The quinone methide analog precursor compound of claim 5, wherein R⁴is a chromophore.
 16. The quinone methide analog precursor compound ofclaim 5, wherein R⁴ is derived from


17. A compound having Formula (VI):

wherein —Z—R¹ is —O—P(O)(OH)₂ or —O-sugar; LG is a leaving group; R³ isa bond, —(CH₂)_(n)NH—, —O(CH₂)_(n)NH—, —N(H)C(O)(CH₂)_(n)NH—,—C(O)N(H)(CH₂)_(n)NH—, —(CH₂)_(n)O—, —O(CH₂)_(n)O—, —O(CH₂CH₂O)_(n)—,—N(H)C(O)(CH₂)_(n)O—, —C(O)N(H)(CH₂)_(n)O—, —C(O)N(H)(CH₂CH₂O)_(n)—,—(CH₂)_(n)S—, —O(CH₂)_(n)S—, —N(H)C(O)(CH₂)_(n)S—, —C(O)N(H)(CH₂)_(n)S—,—(CH₂)_(n)NH—, —C(O)N(H)(CH₂CH₂O)_(n)CH₂CH₂NH,—C(O)(CH₂CH₂O)_(n)CH₂CH₂NH—, —C(O)N(H)(CH₂)_(n)NHC(O)CH(CH₃)(CH₂)_(n)NH—or —N(H)(CH₂)_(n)NH—,

 where each n is independently an integer ranging from 1 to 12; R⁴ is adetectable label; R²¹, R²², R²³, and R²⁴ are each independentlyhydrogen, halo, cyano, aliphatic, alkoxy, NO₂, N(R^(c))₂, aryl,haloalkyl, —C(O)alkyl, —C(S)alkyl, —C(O)OH, —C(O)Oalkyl, —C(O)NHR^(c),—C(O)N(R^(c))₂, —R³R⁴, or two adjacent groups together form an aliphaticring or aryl ring; R^(a) is hydrogen or aliphatic; and each R^(c)independently is hydrogen, aryl, aliphatic or heteroaliphatic.
 18. Thecompound of claim 15, wherein R³—N(H)C(O)(CH₂)_(n)NH—,—C(O)N(H)(CH₂)_(n)NH—(CH₂)_(n)NH—, or —C(O)N(H)(CH₂CH₂O)CH₂CH₂NH—, wheren is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or
 12. 19. The compound of claim15, wherein the detectable label is a chromophore.
 20. The compound ofclaim 15, wherein LG is halo.